Detection and quantification of biomolecules using mass spectrometry

ABSTRACT

The present invention is directed in part to a method for detecting a target nucleic acid using detector oligonucleotides detectable by mass spectrometry. This method takes advantage of the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and releases labels for detection by mass spectrometry. This process is easily incorporated into a polymerase chain reaction (PCR) amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.

RELATED PATENT APPLICATIONS

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 11/950,395, filed Dec. 4, 2007, entitled DETECTIONAND QUANTIFICATION OF BIOMOLECULES USING MASS SPECTROMETRY, naming DirkJohannes Van Den Boom as an inventor, and designated by, which claimedthe benefit of U.S. Provisional Patent Application No. 60/868,718, filedon Dec. 5, 2006, having the same title and named inventor as patentapplication Ser. No. 11/950,395, entitled DETECTION AND QUANTITATION OFBIOMOLECULES USING MASS SPECTROMETRY, naming Dirk Johannes Van Den Boomas an inventor, and designated by. The entirety of each of these patentapplications is incorporated herein by reference.

BACKGROUND

Current methods for detecting and quantifying nucleic acids inmultiplexed assays are limited, especially those assays that utilizefluorescent dyes for detection. For example, White discusses theproblems of multiplexing using the TaqMAN® assay (Trends inBiotechnology (1996) 14(12); 478-483). Among other issues, fluorescentdyes offer only limited multiplexing options, and currently availablemethods that attempt to overcome these limitations, for example, byusing primer-extension and ligation-based SNP analysis followed byuniversal PCR and hybridization to chip arrays are often very timeconsuming (e.g., 1-2 days).

The use of mass spectrometry offers a solution for improved multiplexingbecause of the increased number of detection channels, but the practicalutility of previously disclosed mass spectrometry-based methods can befurther improved. For example, the use of high-specificity hybridizationof peptide nucleic acid (PNA) probes to PCR-amplified DNA and subsequentdetection by mass spectrometry is described by Ross (Anal. Chem (1997)69:4197). Also, a primer extension method and detection of the primerextension product by mass spectrometry is described by Haff (NucleicAcids Res. (1997) 25:3749). Additional mass spectrometry-based methodsare described by Jurinke et al (Adv Biochem Eng Biotechnol (2002)77:57-74).

SUMMARY

The invention in part provides methods for identifying and quantifying atarget biomolecule, such as nucleic acid, and for detecting a targetbiomolecule sequence, such as a nucleic acid sequence or nucleotidesequence. Methods of the invention are advantageous for detectingmultiple target nucleic acids simultaneously in a single sample ormultiple samples while avoiding post-PCR enzymatic processes. Large setsof unique, mass-distinguishable products (MDP's) can be generated thatallow for the simultaneous detection of multiple target nucleic acids.As described further herein, one or more target nucleic acids areamplified by standard amplification methods wherein the amplificationprocess cleaves and degrades detector probes, which yield specificmass-distinguishable products detectable by mass spectrometry. Detectionof multiple MDP's allows for the identification and/or quantification ofmultiple target nucleic acids.

Each mass-distinguishable product (MDP) has a unique physicalcharacteristic that allows it to be uniquely identified when compared toother mass-distinguishable products used in the same assay. Themass-distinguishable products can be separated and identified based onthis difference. For example, MDP's can differ from each other based ontheir unique, predetermined mass and be detected by mass spectrometry.Thus, mass spectrometric analysis reveals the presence of the targetnucleic acid indirectly through the mass-distinguishable product.

The invention therefore in part provides a method of detecting, andoptionally quantifying, a target nucleic acid sequence in anamplification reaction, the method comprising: providing a set ofoligonucleotide primers, wherein a first primer contains a sequencecomplementary to a region in one strand of the target nucleic acidsequence, and a second primer contains a sequence complementary to aregion in a second strand of the target nucleic acid sequence; providingat least one detector oligonucleotide containing a sequencecomplementary to a region of the target nucleic acid, wherein saiddetector oligonucleotide anneals within the target nucleic acid sequencebounded by the oligonucleotide primers in the first step, therebycreating an annealed duplex, and further wherein each oligonucleotideprimer is selected to anneal to its complementary template upstream ofany detector oligonucleotide annealed to the same nucleic acid strand;amplifying the target nucleic acid sequence employing an enzyme having5′ to 3′ nuclease activity as a template-dependent polymerizing agentunder conditions which are permissive for amplification cycling steps of(i) annealing of oligonucleotide primers and detector oligonucleotide toa template nucleic acid sequence contained within the target sequence,and (ii) extending the primer oligonucleotide wherein said nucleic acidamplification enzyme synthesizes a primer extension product while the 5′to 3′ nuclease activity of the nucleic acid amplification enzymesimultaneously releases MDP's from the annealed duplexes comprisingdetector oligonucleotides and its complementary template nucleic acidsequences, thereby creating one or more MDP's; and detecting the one ormore MDP's by mass spectrometry, thereby determining the presence orabsence of the target sequence in a sample. Typically, the amplificationreaction is a polymerase chain reaction. The amplification reaction canalso be a multiplex reaction in which multiple targets are identified.In a related embodiment more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, and all numbers in between, or more target nucleic acidsare detected in a single, multiplexed reaction. In another embodiment,more than one detector oligonucleotide is used to detect more than onetarget nucleic acid in a multiplexed reaction. In another embodiment, aportion of the detector oligonucleotide is complementary to the targetnucleic acid, but it does not need to be perfectly complementary to thetarget nucleic acid. Instead the detector oligonucleotide may besubstantially complementary.

In some embodiments, a plurality of polymorphisms, such as singlenucleotide polymorphisms (SNP's), or genes can be simultaneouslydetermined by combining target nucleic acids with a pair of reagentsunder conditions of target amplification. Each pair of reagents includesan oligonucleotide primer which binds to target nucleic acid and adetector oligonucleotide, which may or may not be modified. In the caseof SNP genotyping, the detector oligonucleotide binds to the site of theSNP and the oligonucleotide has a detection feature that is detectableupon its subsequent release. In a preferred embodiment, the detectionfeature is detectable by mass spectrometry. In the case of geneexpression analysis, the detector oligonucleotide binds to gene-specificsequence and the oligonucleotide has a detection feature that isdetectable upon its subsequent release. The conditions of sequenceamplification can employ a polymerase having 5′-3′ nuclease activity,dNTPs and auxiliary reagents to permit efficient sequence amplification.Examples of auxiliary reagents include, but are not limited to, betain,DMSO for CG-rich regions, detergents, and pyrophosphatases. The sequenceamplification is performed, whereby detector oligonucleotides bound tothe target nucleic acid are cleaved and/or degraded, released andsubsequently detected by mass spectrometry. By having each SNP or geneassociated with a specific MDP, one can determine the SNP's or genespresent in a sample.

In another related embodiment, a polymerase chain reaction (PCR)amplification method of detecting a target nucleic acid sequence in asample is provided, which comprises the steps of: providing to a PCRreaction containing the sample, a set of oligonucleotide primers,wherein a first PCR primer contains a sequence complementary to a regionin one strand of the target nucleic acid sequence, and a second PCRprimer contains a sequence complementary to a region in a second strandof the target nucleic acid sequence; providing at least one detectoroligonucleotide containing a sequence complementary to a region of thetarget nucleic acid, wherein said detector oligonucleotide annealswithin the target nucleic acid sequence bounded by the PCR primers ofthe first step, thereby creating an annealed duplex, and further whereineach PCR primer is selected to anneal to its complementary templateupstream of any detector oligonucleotide annealed to the same nucleicacid strand; amplifying the target nucleic acid sequence employing anucleic acid polymerase enzyme having 5′ to 3′ nuclease activity as atemplate-dependent polymerizing agent under conditions which arepermissive for PCR cycling steps of (i) annealing of primers anddetector oligonucleotide to a template nucleic acid sequence containedwithin the target sequence, and (ii) extending the primer wherein saidnucleic acid polymerase enzyme synthesizes a primer extension productwhile the 5′ to 3′ nuclease activity of the nucleic acid polymeraseenzyme simultaneously releases MDP's from the annealed duplexescomprising detector oligonucleotides and its complementary templatenucleic acid sequences, thereby creating one or more MDP's; anddetecting the one or more MDP's by mass spectrometry, therebydetermining the presence or absence of the target sequence in thesample. In another embodiment, conditions which are permissive for PCRcycling may optionally include denaturation of the strands.

The invention also in part provides a method of detecting a targetnucleic acid, comprising the steps of: annealing an oligonucleotideprimer to the target nucleic acid, annealing a detector oligonucleotideto the same target nucleic acid; introducing an enzyme to extend theoligonucleotide primer in the direction of the detector oligonucleotide,wherein the enzyme cleaves and thereby releases at least a portion ofthe detector oligonucleotide, thereby producing one or more MDP's; anddetecting the one or more MDP's by mass spectrometry. In a relatedembodiment, a portion of the detector oligonucleotide may be partiallycleaved and therefore disassociate from the target nucleic acid. Thus,in this embodiment, the MDP's may include a partially cleaved detectoroligonucleotide that disassociates from the target nucleic acid, and issubsequently detected by mass spectrometry. In another embodiment, asecond oligonucleotide is introduced that binds to the synthesis productof the first oligonucleotide, whereby exponential amplification cansubsequently occur. In a related embodiment, the target nucleic acid isinitially a single-stranded nucleic acid molecule, for example cDNA.

The invention also in part provides non-amplification-based methods ofdetecting a target nucleic acid sequence in a sample, comprising thesteps of: contacting a sample comprising a target nucleic acid with an(i) oligonucleotide primer, comprising a 3′ end and a 5′ end, andcontaining a sequence complementary to a region of the target nucleicacid and a (ii) detector oligonucleotide, comprising a 3′ end and a 5′end, and containing a sequence complementary to a second region of thetarget nucleic acid sequence, thereby creating a (iii) mixture ofduplexes under hybridization conditions, wherein the duplexes comprisethe target nucleic acid annealed to the oligonucleotide primer and tothe detector oligonucleotide such that the 3′ end of the oligonucleotideprimer is upstream of the 5′ end of the detector oligonucleotide;exposing the sample from the first step to a cleavage agent underconditions sufficient to cleave and release the annealed detectoroligonucleotide or at least one fragment thereof, thereby creating oneor more MDP's; and detecting the one or more MDP's by mass spectrometry,thereby detecting the presence or absence of the target nucleic acidsequence in the sample.

The invention further provides, in part, a method of detecting a targetnucleic acid, comprising the steps of: annealing a detectoroligonucleotide to the target nucleic acid; introducing a cleavage agentto cleave at least a portion of the detector oligonucleotide; anddetecting the partially cleaved detector oligonucleotide or fragmentsthereof by mass spectrometry.

In some embodiments, the reaction conditions allow for the extension ofthe oligonucleotide primers, which displaces and degrades the detectoroligonucleotide, thereby yielding oligonucleotide fragments and/or anydetection feature attached thereto. In some embodiments, the targetnucleic acid is amplified prior to the detection method by anamplification reaction. In another embodiment, all of the steps prior todetection are performed simultaneously in a single, closed reactionvessel. In another embodiment, the amplifying or extending step of themethod is repeated until a signal is detected. In a related embodiment,the number of amplifying steps is determined.

In certain embodiments, the detector oligonucleotide is introduced at ahigher concentration relative to the oligonucleotide primer or primers.Also, in some instances, the detector oligonucleotide may benon-extendable by an enzyme. In another embodiment, more than onedetector oligonucleotide is used to detect a single target nucleic acid,or more than one detector oligonucleotide anneals within the targetnucleic acid sequence bounded by the oligonucleotide primers.

In some embodiments, the cleavage agent is an enzyme. In someembodiments, the enzyme has 5′ to 3′ nuclease activity. In otherembodiments the enzyme has polymerase activity capable of amplifyingtarget nucleic acid. In some embodiments, the enzyme is a DNApolymerase, and in other embodiments the enzyme is an RNA dependent DNApolymerase, a DNA dependent RNA polymerase, RNA dependent RNA polymeraseor DNA dependent DNA polymerase. Exemplary DNA polymerases include Taqpolymerase and E. coli DNA polymerase 1. In another embodiment, theenzyme is thermostable.

The invention in part provides for the creation of MDP's resulting fromthe displacement and/or degradation of detector oligonucleotides. In oneembodiment, more than one mass-distinguishable product (MDP) from thesame detector oligonucleotide is detected by mass spectrometry, whichcreates a mass-specific detection signature that corresponds to a targetnucleic acid. In another embodiment, the detector oligonucleotidecomprises a sequence of nucleotides which is non-complementary to thetarget nucleic acid. The non-complementary region may be at the 5′ endof the detector oligonucleotide, in the middle of the detectoroligonucleotide, in which case the detector oligonucleotide is stillcapable of annealing to the target nucleic acid, or at the 3′ end of thedetector oligonucleotide. In other embodiments, the MDP's are capable ofbinding to a solid support upon release (e.g., using streptavidin or acomplementary oligonucleotide attached to a bead). For example, theMDP's may bind directly to a matrix for MALDI-TOF mass spectrometryanalysis. In another embodiment, the one or more MDP's are amplifiedafter their release, for example, by using a universal primer system.

In some embodiments, detector oligonucleotides are incorporated into anucleic acid detection method that utilizes universal primers to amplifysequence-specific primers or oligonucleotides resulting from enzymaticmodifications, wherein the amplification process yieldsmass-distinguishable products detectable by mass spectrometry. Morespecifically, an embodiment of the invention includes providing aplurality of target nucleic acid sequences each comprising from 3′ to 5′a first, second and third target domain, the first target domaincomprising a detection position, the second target domain being at leastone nucleotide, contacting the target nucleic acid sequences with setsof probes for each target sequence, each set comprising: a first probecomprising from 5′ to 3′, a first domain comprising a first universalpriming sequence, a second domain comprising a detector oligonucleotidebinding domain and a third domain comprising a sequence substantiallycomplementary to the first target domain of a target sequence, and aninterrogation position within the 3′ four terminal bases, a second probecomprising a first domain comprising a sequence substantiallycomplementary to the third target domain of a target sequence, to form aset of first hybridization complexes, and a second domain comprising asecond universal priming sequence, contacting the first hybridizationcomplexes with at least a first universal primer that hybridize to thefirst universal priming sequence, an extension enzyme and dNTPs, underconditions whereby if the base at the interrogation positions arecomplementary with the bases at the detection positions, extension ofthe first probes occurs through the second target domains to form secondhybridization complexes, contacting the second hybridization complexeswith a ligase to ligate the extended first probes to the second probesto form amplification templates. The embodiment further includesintroducing detector oligonucleotides, wherein specific detectoroligonucleotides anneal to the sequence-specific amplificationtemplates, introducing an enzyme to amplify the amplification templates,and detecting the one or more MDP's resulting from the amplificationreactions, wherein determining the presence or absence of the targetnucleic sequences. In another embodiment, the methods of the presentinvention are utilized in conjunction with the methods disclosed in U.S.Pat. No. 6,797,470; U.S. Pat. No. 6,890,741, U.S. Pat. No. 6,812,005,U.S. Pat. No. 6,890,741, US Patent Application Publication No.20020006617, US Patent Application Publication No. 20030036064, USPatent Application Publication No. 20030104434, US Patent ApplicationPublication No. 20030211489, US Patent Application Publication No.20030108900, US Patent Application Publication No. 20030170684, USPatent Application Publication No. 20040121364, US Patent ApplicationPublication No. 20040224352, US Patent Application Publication No.20040224352, all of which are hereby incorporated by reference.

In some embodiments, the detector oligonucleotide is not modified, inwhich case unmodified MDP's comprising oligonucleotide fragments aredetected by mass spectrometry. In other embodiments, the detectoroligonucleotide comprises one or more nucleoside modifications.Nucleoside modifications include modifications to a nucleotide,phosphate backbone or sugar moiety. The nucleoside modification mayoccur in a non-complementary region of the detector oligonucleotide, atthe 5′ end of the detector oligonucleotide, at the 3′ end of thedetector oligonucleotide, or in the middle of the detectoroligonucleotide, which ensures target-specific hybridization of thedetector oligonucleotide to the target nucleic acid. In anotherembodiment, the nucleoside modification is selected from the groupconsisting of isotopic enrichment, isotopic depletion and halogenmodification. In another embodiment, isotopic coding is achieved by theintroduction of deuterium, or other suitable isotopes.

In certain embodiments, the detector oligonucleotide comprises one ormore cleavage recognition sites. In another embodiment, the detectoroligonucleotide comprises one or more non-degradable nucleotides. In yetanother embodiment, the detector oligonucleotide comprises one or morecleavage recognition sites and one or more non-degradable nucleotides.

In a preferred embodiment, the detector oligonucleotide comprises one ormore locked nucleic acids (LNAs), which serve as non-degradablenucleotides and thereby control the point of cleavage. LNAs bind verystably with their complement and have a highly reduced rate of cleavagerelative to a nascent deoxynucleotide. This effect may be furtherenhanced by placing two or LNAs adjacent to each other. Additionally,LNAs increase the melting temperature of the oligonucleotides of whichthey are incorporated. The cleavage site for the mass degradationproducts are thus controlled for predictability of product size andproper identification. Linked nucleic acids sometimes are referred to as“inaccessible RNA,” as such nucleotides often are modified RNAnucleotides. The ribose moiety of an LNA nucleotide often is modifiedwith an extra bridge connecting the 2′ and 4′ carbons. The bridge“locks” the ribose in the 3′-endo structural conformation, which isoften found in the A-form of DNA or RNA. LNA nucleotides can be mixedwith DNA and/or RNA bases in an oligonucleotide whenever desired, andsuch oligomers are commercially available. The locked riboseconformation enhances base stacking and backbone pre-organization, whichsignificantly increases the thermal stability (melting temperature) ofoligonucleotides.

Additionally, the cleavage site for the detector oligonucleotide may becontrolled and/or inhibited by the use of abasic nucleotides (e.g.,1′,2′-Dideoxyribose).

In another embodiment, the detector oligonucleotide comprises one ormore peptide nucleic acids (PNAs) or nucleic acid peptide conjugates.

In some embodiments, the detector oligonucleotide comprises one or moredetection moieties. In one embodiment, the detection moiety may be anyone or more of a compomer, sugar, peptide, protein, antibody, chemicalcompound (e.g., biotin), mass tag (e.g., metal ions or chemical groups),fluorescent tag, charge tag (e.g., such as polyamines or charged dyes)and hydrophobic tag. In a related embodiment, the detection moiety is amass-distinguishable product (MDP) or part of an MDP detected by massspectrometry. In a specific embodiment, the detection moiety is afluorescent tag or label that is detected by mass spectrometry. In someembodiments, the detection moiety is at the 5′ end of the detectoroligonucleotide, the detection moiety is attached to a non-complementaryregion of the detector oligonucleotide, or the detection moiety is atthe 5′ terminus of the non-complementary sequence. In anotherembodiment, the detection moiety is incorporated into or linked to aninternal nucleotide or to a nucleotide at the 3′ end of the detectoroligonucleotide. In yet another embodiment, one or more detectionmoieties are used either alone or in combination.

In certain embodiments, the detection moiety is a synthetic polymer or abiopolymer or some combination thereof, while in other embodiments, thedetection moiety is any compound that may be detected by massspectrometry. In particular embodiments, the detection moiety is abiopolymer comprising monomer units, wherein each monomer unit isseparately and independently selected from any one or more of an aminoacid, a nucleic acid, and a saccharide. Amino acids and nucleic acidsare the preferred monomer units. Because each monomer unit may beseparately and independently selected, biopolymer detection moieties maybe polynucleic acids, peptides, peptide nucleic acids, oligonucleotides,and so on.

In some embodiments, the detection moiety is a synthetic polymer, suchas polyethylene glycol, polyvinyl phenol, polypropylene glycol,polymethyl methacrylate, and derivatives thereof. Synthetic polymers maytypically contain monomer units selected from the group consistingessentially of ethylene glycol, vinyl phenol, propylene glycol, methylmethacrylate, and derivatives thereof. More typically the detectionmoiety may be a polymer containing polyethylene glycol units.

The invention in part provides detector oligonucleotides that serve asprobes that bind to specific target nucleic acid sequences. In anembodiment, the detector oligonucleotide selectively binds to agene-specific or genome-specific sequence, which thereby allows for geneexpression or copy number variant analysis. In another embodiment, thedetector oligonucleotide selectively binds to an allele-specificsequence. In a related embodiment, the allele-specific nucleotide baseor bases of the detector oligonucleotide comprises a detection moiety ora nucleoside modification. In a preferred embodiment, theallele-specific nucleotide base or bases of the detector oligonucleotidefall in the middle or towards the 5′ or 3′ end of the detectoroligonucleotide.

The invention also in part provides methods of detecting target nucleicacids based on epigenetic differences, such as methylation, acetylationand other non-sequence altering modifications. In one embodiment, thedetector oligonucleotide selectively binds to a methylation-specificsequence based on the methylation status of the target nucleic acid. Ina related embodiment, the detector oligonucleotide selectively binds toa methylation-specific sequence based on the methylation status of thetarget nucleic acid prior to bisulfite treatment, or, alternatively,after bisulfite treatment. In another embodiment, target nucleic acidsare selectively enriched for methylated DNA (either before amplificationor after amplification) by coating a container with a polypeptidecapable of binding methylated DNA; contacting said polypeptide with asample comprising methylated and/or unmethylated DNA; and detecting thebinding of said polypeptide to methylated DNA. Methods for detectingmethylated DNA are described in PCT Patent Publication No. WO06056478A1,which is hereby incorporated by reference.

The present invention in part provides methods for detecting a targetnucleic acid. Generally, the method includes obtaining a plurality ofdetector oligonucleotides, each detector oligonucleotide designedspecifically for a given assay (e.g., allele-specific, gene-specific,sequence-specific, methylation-specific, etc.), as described above. Itis preferred that each detector oligonucleotide within the plurality iscapable of yielding one or more unique mass-distinguishable productsthat correlate with the presence or absence of the target nucleic acid.By “unique mass-distinguishable product” it is meant that each detectoroligonucleotide within the plurality will yield differentmass-distinguishable product(s) from all other detector oligonucleotidesin the plurality. A plurality will generally be understood to includetwo or more detector oligonucleotides. Next, the target molecule iscontacted with the plurality of detector oligonucleotides underconditions suitable to allow for the generation of MDP's, which areanalyzed by mass spectrometry. Typically, the mass is indicative of aspecific target nucleic acid. In this way, the target molecule can beidentified according to the unique combination of MDP's. Example 1provides an example of a sequence-specific assay for the detection ofexon 10 of the Rhesus D gene. In another example, an assay is designedfor each particular SNP of a target nucleic acid, wherein the detectoroligonucleotide of the assay yields unique MDP's depending on which SNPis present. If two SNP's are to be detected at a particular position,two allele-specific detector oligonucleotides with unique MDP's areused. For gene expression analysis, a gene-specific detectoroligonucleotide and a competitor may be used. See, for example, USPatent Application No. 20040081993 (Cantor et al.), which is herebyincorporated by reference.

In some embodiments, the oligonucleotide primer selectively binds to anallele-specific sequence. In some embodiments, the 3′ end of theoligonucleotide primer is at least one base upstream of the 5′ end ofthe detector oligonucleotide.

The invention in part provides methods of detecting MDP's by massspectrometry. In an embodiment, the detection is done by a massspectrometer, which may be one of the following: MALDI-TOF MS, TandemMS, ESI-TOF, ESI-iontrap, LC-MS, GC-MS, ion mobility MS, laserdesorption ionization mass spectrometry (LDI-MS) and quadrupole-MS.Other mass spectrometry devices and methods now existing or which may bedeveloped are within the scope of the present invention.

The invention also in part provides methods of detecting and quantifyingbiomolecules, such as target nucleic acids, wherein the generation ofPCR product is monitored by detection of mass-distinguishable product(MDP). In one embodiment, the detection is done in real-time. In someembodiments, the detection in real-time is performed with anelectrospray mass spectrometer or LC-MS. In another embodiment, the oneor more MDP's are spotted at specific locations on a massspectrometry-related medium that corresponds to a specific time duringthe amplification process. An example of a mass spectrometry-relatedmedium is a matrix suitable for MALDI-TOF MS. In another embodiment, acompetitor template nucleic acid is introduced, wherein the templatenucleic acid serves as an internal control. In yet another embodiment,the number of amplification cycles is determined to obtain aquantitative result. The amount of starting target nucleic acid presentin the reaction mixture may be quantified by cycle threshold (Ct), orany other method known in the art.

Also provided herein are methods for detecting a target nucleic acidsequence, which comprise analyzing a nucleic acid sample containingmass-distinguishable products by mass spectrometry, wherein themass-distinguishable products result from (a) annealing anoligonucleotide primer to a target nucleic acid; (b) annealing adetector oligonucleotide to the same target nucleic acid; and (c)contacting the target nucleic acid with an enzyme that extends theoligonucleotide primer in the direction of the detector oligonucleotide,wherein: the detector oligonucleotide or portion thereof iscomplementary to the target nucleic acid sequence, and the enzymecleaves and thereby releases at least a portion of the detectoroligonucleotide, thereby producing one or more mass-distinguishableproducts; whereby the target nucleic acid sequence is detected byidentifying the mass-distinguishable products by mass spectrometry. Incertain embodiments, a second oligonucleotide is introduced that bindsto the synthesis product of the first oligonucleotide, wherebyexponential amplification can subsequently occur.

Provided also are methods for detecting a target nucleic acid sequence,which comprises analyzing a nucleic acid sample containingmass-distinguishable products by mass spectrometry, wherein themass-distinguishable products result from (a) contacting a targetbiomolecule with a detectable probe containing an oligonucleotide thatserves as a template nucleic acid under conditions in which thedetectable probe specifically binds to the target biomolecule; (b)annealing an oligonucleotide primer to the template nucleic acid; (c)annealing a detector oligonucleotide to the same template nucleic acid;and (d) contacting the template nucleic acid with an enzyme that extendsthe oligonucleotide primer in the direction of the detectoroligonucleotide, wherein: the detector oligonucleotide or portionthereof is complementary to the target nucleic acid sequence, and theenzyme cleaves and thereby releases at least a portion of the detectoroligonucleotide, thereby producing one or more mass-distinguishableproducts; whereby the target nucleic acid sequence is detected byidentifying the mass-distinguishable products by mass spectrometry. Incertain embodiments, a second oligonucleotide is introduced that bindsto the synthesis product of the first oligonucleotide, wherebyexponential amplification can subsequently occur.

Detector oligonucleotides may be part of a library that are designed toyield a range of mass distinguishable products (or species) that can bedetected individually or as part of a multiplexed reaction. The libraryof detector oligonucleotides may be designed, including theincorporation of modifications, to improve certain characteristics. Suchcharacteristics include without limitation, binding specificity to thetarget nucleic acid, generating uniform cleavage products and/or havingdetection properties (e.g., mass) of mass distinguishable products(MDPs) that improve multiplexing capabilities. In one aspect, thelibrary comprises two or more detector oligonucleotide species, whereeach species comprises a 3′ portion that is complementary orsubstantially complementary to the target nucleic acid and a 5′ portionthat is susceptible to cleavage by a cleavage agent, and therebyproduces a mass distinguishable product (MDP) that is unique to eachdetector oligonucleotide species and each corresponding target nucleicacid. In certain embodiments, the complementary portion of a detectoroligonucleotide species is modified to include one or more modificationssuch as minor groove binding moieties and locked nucleic acids (LNA's).In some embodiments, the non-complementary 5′ portion of a detectoroligonucleotide species is modified to include one or moremodifications, including without limitation, abasic nucleotides, acapture mechanism (e.g., biotin or an oligonucleotide complementary to acapture probe (but not the target nucleic acid)), a releasing mechanismfor subsequent release after capture (e.g., photocleavable linker) andmass modifiers. Thus, in certain embodiments, the invention in partpertains to a detector oligonucleotide comprising a complementaryportion that is complementary to a target nucleic acid, and anon-complementary portion that is susceptible to cleavage by a cleavageagent, thereby producing a mass distinguishable product (MDP). In someembodiments, the detector oligonucleotide comprises a modificationselected from the group consisting (i) a modification that increases themelting temperature of the detector oligonucleotide, (ii) a modificationthat increases resistance of the detector oligonucleotide to cleavage,(iii) a modification that makes the 5′ portion of the detectoroligonucleotide less likely to hybridize to the target nucleic acid thanwithout the modification, (iv) a modification of incorporating a capturemechanism in the detector oligonucleotide, (iv) a modification ofincorporating a releasing mechanism in the detector oligonucleotide. Incertain embodiments, the complementary portion comprises a modificationthat increases the melting temperature of the detector oligonucleotide,and sometimes the modification is the incorporation of one or more minorgroove binders to the detector oligonucleotide. In some embodiments, thecomplementary portion comprises a modification that increases resistanceof the detector oligonucleotide to cleavage, and sometimes themodification is the incorporation of one or more locked nucleic acids(LNAs) or amadites. In certain embodiments, the non-complementaryportion comprises a modification that makes the 5′ portion of thedetector oligonucleotide less likely to bind to the target nucleic acidwithout said modification, and sometimes the modification is theincorporation of one or more abasic nucleotides. In some embodiments,the non-complementary portion comprises a capture mechanism that allowsfor the mass-distinguishable product (MDP) to bind to a solid supportupon its cleavage from the detector oligonucleotide by a cleavage agent,and sometimes the capture mechanism is a biotin molecule. In certainembodiments, the mass-distinguishable product is amplified after itsrelease from the detector oligonucleotide. In some embodiments, thedetector oligonucleotide comprises a universal primer hybridization siteand is amplified using a universal primer. In certain embodiments, thedetector oligonucleotide comprises one or more releasing mechanisms forsubsequent release of the MDP or fragment thereof after the MDP orfragment thereof binds to the solid support, and sometimes the one ormore releasing mechanisms are selected from the group consisting ofenzymatic cleavage, photocleavage, exchange reactions andtemperature-sensitive release. In some embodiments, thenon-complementary portion comprises a detection moiety. In certainembodiments, the detector oligonucleotide comprises two or more, orthree or more, of the modifications selected from the group consistingof minor groove binders, locked nucleic acids, amadites, abasicnucleotides, capture mechanism, release mechanism and detection moiety.

An aspect of the invention also pertains to a library comprising two ormore detector oligonucleotides described herein, where each massdistinguishable product corresponds to a unique target nucleic acid. Incertain embodiments, the library comprises 10 or more detectoroligonucleotide species, 50 or more detector oligonucleotide species,100 or more detector oligonucleotide species or 250 or more detectoroligonucleotide species.

Certain features of embodiments of the invention are described ingreater detail in the detailed description, claims and drawings thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating detection of a target nucleic acidusing mass spectrometry. The reaction components include a targetnucleic acid, forward and reverse PCR primers, a detectoroligonucleotide, an amplification enzyme, and amplification reagentssuch as buffer(s) and nucleotides. In step A, extension of the primersoccurs. During extension, the detector oligonucleotide is displaced anddegraded (step B). During each amplification cycle, mass-distinguishableproducts (MDP's) (also referred to as degradation products in theFigure) are generated by the 5′ nuclease activity of the enzyme (stepC). Following amplification, MDP's may be optionally conditioned, andlater detected by mass spectrometry. Reaction byproducts may include,inter alia, PCR product, leftover primers, undegraded oligonucleotideprimers and MDP's (step D). Step E shows an exemplary spectrogram, wherethe y-axis is arbitrary intensity (a.i.) and the x-axis is mass (m) overz (charge). The presence of the MDP's confirms the presence of targetnucleic acid.

FIG. 2 is a schematic illustrating detection of a target nucleic acidusing mass spectrometry, wherein the detector oligonucleotide comprisesa 5′ non-complementary region. In step B, the enzyme releases the 5′non-complementary region of the detector oligonucleotide, and it isdetected in step C. FIG. 9 provides an experimental spectrogram where a5′ non-complementary MDP is generated and detected. The term “identifiestarget NS” at the bottom of the Figure refers to “identifies targetnucleotide sequence.”

FIG. 3 is a schematic illustrating detection of a target nucleic acidusing mass spectrometry, wherein the detector oligonucleotide comprisesa 3′ non-complementary region. In step B, the enzyme releases the 3′non-complementary region of the detector oligonucleotide, and it isdetected in step C. The term “identifies target NS” at the bottom of theFigure refers to “identifies target nucleotide sequence.” In oneembodiment the invention in part may include a 3′ non-complementaryregion that is cleaved and detected. In a further related embodiment thehybridized oligonucleotides upstream of the 3′ non-complementary regionare non-cleavable or non-degradable or abasic in nature, therebyproducing a unique defined cleavage product.

FIG. 4 is a schematic illustrating a multiplexed assay for detectingmultiple target nucleic acids. An assay is designed for each target ofthe four targets (1-4), where each target has a unique detectoroligonucleotide with a 5′ non-complementary region of a specific length(L1-L4). Upon amplification (step A), the 5′ non-complementary regionMDP is generated if the target is present. In this particular example,targets 1 and 4 are present, so L1 and L4 MDP's are generated anddetected by mass spectrometry (as illustrated in the spectrogram of stepB).

FIG. 5 is a schematic illustrating a real-time embodiment of theinvention. The scheme is similar to that shown in FIG. 1, however, massspectrometry analysis is performed after every cycle or at given timepoints (e.g., after every 5 cycles). If target nucleic acid is present,the degradation product (i.e., MDP) signal intensity increases withevery cycle while the primer and undegraded detector oligonucleotidesignal intensity decreases due to consumption and degradation.

FIG. 6 is a schematic illustrating a variation of an assay in whichuniversal primers are utilized to detect a target biomolecule. In thisparticular figure, a genotyping assay is shown where each singlenucleotide polymorphism (SNP) has a unique set of primers. For example,SNP3 is assayed using allele-specific primers with sequence regions 5and 6 (S5 and S6) corresponding to the C allele and G allele,respectively. A downstream primer is also introduced, designated CS3, orcommon primer3. In step A, the primers hybridize to the target, andallele-specific primer extension occurs if the allele is present. Uponextension, a ligation product forms. Detector oligonucleotidescomplementary to the assay-specific sequence regions of the ligationproduct are introduced, designated cS5 and cS6, or complementary primer5and 6 for the SNP3 assay. Upon amplification of the ligation productusing universal primers, the detector oligonucleotides (cS5 and cS6) aredisplaced and degraded to yield MDP's, which are detected by massspectrometry. The exemplary mass spectrogram reveals the presence ofallele A for SNP1, allele T for SNP2, and both alleles C and G(heterozygous) for SNP 3.

FIG. 7 is a schematic illustrating an embodiment of the invention usefulfor non-nucleic acid detection (e.g., protein detection). Each aptamercontains a target binding domain (A1-A3), a unique sequence (S) to whicha unique complementary detector oligonucleotide (CS) can bind duringsubsequent amplification, and universal primer binding sites. In step A,target proteins bind to immobilized antibodies. In step B, non-bindingreagents are removed by washing, and an aptamer library is added.Complementary aptamers bind to target proteins, and non-complementaryaptamers are washed away. Universal primers and sequence-specificdetector oligonucleotides are added (step C). Upon amplification, thedetector oligonucleotides are displaced and degraded to yield MDP's,which are detected by mass spectrometry (step D). The presence of MDP1and MDP3 indicate the presence of protein1 and protein3, respectively.

FIG. 8 is a mass spectrogram showing the MDP's generated from the Exon10specific detector oligonucleotide during PCR amplification (mass rangebetween 1900 and 3000 Da). The mass signals at 2123.6 Da, 2437.0 Da and2726.2 Da represent 5′ MDP's containing the polyA tag (6 Adenine)cleaved at the first hybridized T nucleotide (AAAAAAT), the polyA tagcleaved after the first two hybridized nucleotides (AAAAAATA) and afterthe first three hybridized nucleotides (AAAAAATAC). The Y-axis is signalintensity and the X-axis is mass to charge ratio.

FIG. 9 is a mass spectrogram showing the MDP's generated from the Exon5specific detector oligonucleotide generated during PCR amplification(mass range between 2500 and 7000 Da). The mass signals at 5765 and6335.9 Da represent remaining, unused PCR primer. The mass signals at2741.6 Da and 3032.6 Da represent 5′ MDP's containing the polyA tag (8Adenine) cleaved at the first hybridized T nucleotide (AAAAAAAAT) andthe polyA tag cleaved after the first two hybridized nucleotides(AAAAAATC). The Y-axis is signal intensity and the X-axis is mass tocharge ratio.

DETAILED DESCRIPTION

The present invention offers several advantages over current nucleicacid detection and quantification methods, such as increasedmultiplexing, assay simplicity, fast cycling times and no post PCRprocessing, for example. Current methods often require multi-stepreactions including solid-phases purifications, transferring and washingsteps, and post-PCR enzymatic reactions, all of which increase the totalassay time and cost, thereby limiting the methods applicability. Forexample, TaqMan-based QPCR methods are limited by their use of dyes,whereas the present method is only limited by the number of uniquedetection features designed for each assay. This allows for theunambiguous detection of MDP's across a finite mass range.

The assay is also simple. In one embodiment, there is only oneamplification or extension step, which occurs in a closed tube, soproducts are not transferred or subjected to a post-amplificationenzymatic reaction. Also, the amplification step allows for fast cycling(i.e., the assay finishes in the plateau phase), so the speed and turnaround time is only limited by the cycling speed—and fast cycles can beused. Finally, there is no post-amplification processing. For example,none of the products need to be captured on a solid support or bound toa capture probe and further manipulated by enzymes.

The invention provides an advantage of simultaneously identifying andoptionally quantifying large numbers of sequences from one or moresamples for a range of applications, including, but not limited to,diagnostics, forensics and security applications such as identificationof individuals (e.g., airline passengers). In certain applications, theinvention provides the advantage of analyzing one or more samples forthe presence or absence of multiple polymorphisms associated with aparticular disease or diseases, to analyze the expression of one or moregenes associated with a particular disease or diseases, or to identifythe origin of one or more samples in a simple, fast multiplexed assaythat can be done in hours rather than days.

Methods of the invention find utility in performing multiplexed assaysfor detection/analysis of biomolecule targets including, but not limitedto nucleic acid detection, such as sequence recognition, SNP detection,transcription analysis or mRNA determination, allelic determination,mutation determination and methylation analysis. In another embodiment,the methods of the present invention may be used in combination with aproximity ligation or immunoassay-based method for the detection andquantification of non-nucleic acid biomolecules, such as proteins orpeptides. For example, in one embodiment, a detector oligonucleotide ofthe invention is annealed to a ligated complex generated during anearlier proximity ligation reaction or immunoassay, which subsequentlyserves as a template for nucleic acid amplification reactions. Uponamplification of the ligation complex, mass-distinguishable products arecreated and detected as described herein, thus allowing for increasedmultiplexing. Proximity ligation and immunoassays are described furtherin U.S. Pat. No. 5,665,539; U.S. Pat. No. 6,511,809; U.S. Pat. No.6,878,515; US Patent Application No. 20050233351; US Patent ApplicationNo. 20020064779; and by Roger Brent and his colleagues at the MolecularSciences Institute (Berkeley, Calif.) in. Nat. Methods. 2005 January;2(1):31-7, all of which are hereby incorporated by reference.

An advantage to using an amplification-based method that generatesmass-distinguishable products detectable by mass spectrometry methods isthe ability to simultaneously detect many target nucleic acids at thesame time. Present methods are limited due to broad overlappingspectrums produced by existing fluorescent chromophore-based methods.Therefore, an upper limit for fluorescence multiplexing is most likelyto be about ten different labels. Present mass spectrometry-basedmethods are useful for multiplexed reactions up to about 50-plexes. Withthe mass spectrometry-based method disclosed herein, multiplexing ofgreater than about fifty or hundreds, and perhaps even thousands, ofdifferent targets is possible. Due to this high level multiplexingability, not only can many assays be used at the same time, anyindividual detector oligonucleotide can be labeled with many differentdetection features.

Finally, the assay has tremendous utility for any field that requiresfast turnaround and multiplexing capabilities (e.g., public security).The assay is fast and robust, and the detection platform is small,highly accurate and easy-to-use, which makes the methods of the presentinvention ideal for a wide range of applications, including detection ofinfectious agents within a clinical sample, forensics, diagnostics,research (e.g., detection of a gene (cDNA) insert within a clone),security and field use.

A. DEFINITIONS

The term “sample” as used herein includes a specimen or culture (e.g.,microbiological cultures) that includes nucleic acids. The term “sample”is also meant to include both biological and environmental samples. Asample may include a specimen of synthetic origin. Biological samplesinclude whole blood, serum, plasma, umbilical cord blood, chorionicvilli, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid(e.g., bronchioalveolar, gastric, peritoneal, ductal, ear,arthroscopic), biopsy sample, urine, feces, sputum, saliva, nasalmucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat,breast milk, breast fluid, embryonic cells and fetal cells. In apreferred embodiment, the biological sample is blood, and morepreferably plasma. As used herein, the term “blood” encompasses wholeblood or any fractions of blood, such as serum and plasma asconventionally defined. Blood plasma refers to the fraction of wholeblood resulting from centrifugation of blood treated withanticoagulants. Blood serum refers to the watery portion of fluidremaining after a blood sample has coagulated. Environmental samplesinclude environmental material such as surface matter, soil, water andindustrial samples, as well as samples obtained from food and dairyprocessing instruments, apparatus, equipment, utensils, disposable andnon-disposable items. These examples are not to be construed as limitingthe sample types applicable to the present invention.

The terms “target” or “target nucleic acid” as used herein are intendedto mean any molecule whose presence is to be detected or measured orwhose function, interactions or properties are to be studied. Therefore,a target includes essentially any molecule for which a detectable probe(e.g., detector oligonucleotide) or assay exists, or can be produced byone skilled in the art. For example, a target may be a biomolecule, suchas a nucleic acid molecule, a polypeptide, a lipid, or a carbohydrate,that is capable of binding with or otherwise coming in contact with adetectable probe (e.g., an antibody), wherein the detectable probe alsocomprises nucleic acids capable of being detected by methods of theinvention. As used herein, “detectable probe” refers to any molecule oragent capable of hybridizing or annealing to a target biomolecule ofinterest and allows for the specific detection of the target biomoleculeas described herein. In one aspect of the invention, the target is anucleic acid, and the detectable probe is a detector oligonucleotide.The terms “nucleic acid” and “nucleic acid molecule” may be usedinterchangeably throughout the disclosure. The terms refer tooligonucleotides, oligos, polynucleotides, deoxyribonucleotide (DNA),genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA),bacterial DNA, viral DNA, viral RNA, RNA, message RNA (mRNA), transferRNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones,plasmids, M13, P1, cosmid, bacteria artificial chromosome (BAC), yeastartificial chromosome (YAC), amplified nucleic acid, amplicon, PCRproduct and other types of amplified nucleic acid, RNA/DNA hybrids andpolyamide nucleic acids (PNAs), all of which can be in either single- ordouble-stranded form, and unless otherwise limited, would encompassknown analogs of natural nucleotides that can function in a similarmanner as naturally occurring nucleotides and combinations and/ormixtures thereof. Thus, the term “nucleotides” refers to bothnaturally-occurring and modified/nonnaturally-occurring nucleotides,including nucleoside tri, di, and monophosphates as well asmonophosphate monomers present within polynucleic acid oroligonucleotide. A nucleotide may also be a ribo; 2′-deoxy; 2′,3′-deoxyas well as a vast array of other nucleotide mimics that are well-knownin the art. Mimics include chain-terminating nucleotides, such as3′-O-methyl, halogenated base or sugar substitutions; alternative sugarstructures including nonsugar, alkyl ring structures; alternative basesincluding inosine; deaza-modified; abasic nucleotides, such as1′,2′-Dideoxyribose; chi, and psi, linker-modified; mass label-modified;phosphodiester modifications or replacements including phosphorothioate,methylphosphonate, boranophosphate, amide, ester, ether; and a basic orcomplete internucleotide replacements, including cleavage linkages sucha photocleavable nitrophenyl moieties.

The presence or absence of a target can be measured quantitatively orqualitatively. Targets can come in a variety of different formsincluding, for example, simple or complex mixtures, or in substantiallypurified forms. For example, a target can be part of a sample thatcontains other components or can be the sole or major component of thesample. Therefore, a target can be a component of a whole cell ortissue, a cell or tissue extract, a fractionated lysate thereof or asubstantially purified molecule. Also a target can have either a knownor unknown sequence or structure.

The term “amino acid” as used herein refers to naturally-occurring aminoacid as well as any modified amino acid that may be synthesized orobtained by methods that are well known in the art.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification. Components of an amplificationreaction may include, but are not limited to, e.g., primers, apolynucleotide template, polymerase, nucleotides, dNTPs and the like.The term “amplifying” typically refers to an “exponential” increase intarget nucleic acid. However, “amplifying” as used herein can also referto linear increases in the numbers of a select target sequence ofnucleic acid, but is different than a one-time, single primer extensionstep.

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990.

“Oligonucleotide” as used herein refers to linear oligomers of naturalor modified nucleosidic monomers linked by phosphodiester bonds oranalogs thereof. Oligonucleotides include deoxyribonucleosides,ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs),and the like, capable of specifically binding to a target nucleic acid.Usually monomers are linked by phosphodiester bonds or analogs thereofto form oligonucleotides ranging in size from a few monomeric units,e.g., 3-4, to several tens of monomeric units, e.g., 40-60. Whenever anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′-3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, “T” denotes deoxythymidine,and “U” denotes the ribonucleoside, uridine, unless otherwise noted.Usually oligonucleotides comprise the four natural deoxynucleotides;however, they may also comprise ribonucleosides or non-naturalnucleotide analogs. Where an enzyme has specific oligonucleotide orpolynucleotide substrate requirements for activity, e.g., singlestranded DNA, RNA/DNA duplex, or the like, then selection of appropriatecomposition for the oligonucleotide or polynucleotide substrates is wellwithin the knowledge of one of ordinary skill.

As used herein “oligonucleotide primer”, or simply “primer”, refers to apolynucleotide sequence that hybridizes to a sequence on a targetnucleic acid template and facilitates the detection of a detectoroligonucleotide. In amplification embodiments of the invention, anoligonucleotide primer serves as a point of initiation of nucleic acidsynthesis. In non-amplification embodiments, an oligonucleotide primermay be used to create a structure that is capable of being cleaved by acleavage agent. Primers can be of a variety of lengths and are oftenless than 50 nucleotides in length, for example 12-25 nucleotides, inlength. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art.

The term “detector oligonucleotide” as used herein refers to apolynucleotide sequence capable of hybridizing or annealing to a targetnucleic acid of interest and allows for the specific detection of thetarget nucleic acid.

A “mismatched nucleotide” or a “mismatch” refers to a nucleotide that isnot complementary to the target sequence at that position or positions.A detector oligonucleotide may have at least one mismatch, but can alsohave 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides.

The methods and compositions of the invention can be used to detectand/or quantify any genetic or genomic sequence, variation orrearrangement such as polymorphisms (e.g., SNPs or CNPs),insertion/deletions, splice-variants, aneuploidies, loss ofheterozygosity, copy number variants. The methods and compositions mayalso be used for single molecule copy detection (for example using“Digital PCR”) or any sequence-specific feature. The term “polymorphism”as used herein refers to an allelic variant. Polymorphisms can includesingle nucleotide polymorphisms (SNP's) as well as simple sequencelength polymorphisms. A polymorphism can be due to one or morenucleotide substitutions at one allele in comparison to another alleleor can be due to an insertion or deletion, duplication, inversion andother alterations known to the art.

The term “mass-distinguishable product” as used herein may be usedinterchangeably with “cleavage product”, “degradation product” or “probefragment”. In addition the acronym “MDP” may be used. The term“mass-distinguishable product” refers to the one or more degradationproducts resulting from the cleavage and release of the detectoroligonucleotide as described by the methods herein. Themass-distinguishable products (MDP's) may include, but are not limitedto, unmodified detector oligonucleotide fragments, modified detectoroligonucleotide fragments (e.g., isotopically enriched or depletedoligonucleotide fragments), oligonucleotide fragments comprisingdetection moieties, detection moieties released from the detectoroligonucleotide, and detector oligonucleotide fragments notcomplementary to the target nucleic acid. The mass-distinguishableproducts may also include partially cleaved detector oligonucleotidesfragments that disassociate from the target nucleic acid upon partialdegradation of the detector oligonucleotide.

The term “mass-specific detection signature” as used herein refers tothe instance when more than one mass-distinguishable product isdetected, thus resulting in a spectrogram with more than one mass peakper target nucleic acid. Upon cleavage of the detector oligonucleotide,multiple MDP's may be generated that are detectable by massspectrometry. Each detection assay may have its own mass-specificdetection signature comprising multiple cleavage products with differentmasses that correspond to the same target nucleic acid.

The term “modified” as used herein refers to a detector oligonucleotidethat has been altered to include a detection feature.

The term “detection feature” as used herein refers to a modificationthat has been introduced to create a separation characteristic that isdetectable, for example by a mass difference or a size difference, bymass spectrometry or any other size-based separation method such as gelelectrophoresis (on a variety of supports including acrylamide oragarose gels, paper, etc.), chromatography or filtration. Separationcharacteristics allow for the detection of a specific MDP or subset ofMDP's from a larger set of MDP's. Detection features include, but arenot limited to, detection moieties and nucleoside modifications.

The term “detection moiety” as used herein refers to any atom ormolecule that can be used to provide a detectable (preferablyquantifiable) effect and that can be attached to or incorporated into anucleic acid (e.g., detector oligonucleotide). In one preferredembodiment, the detection moiety is a moiety characterized by a uniquemass, allowing specific identification in a mass-based separation, e.g.,by mass spectrometry, gel electrophoresis, chromatography or filtration.Detection moieties include, but are not limited to, nucleotides,compomers, sugars, peptides, nucleotide-peptide conjugates, proteins andantibodies, chemical compounds, metal compounds, electron-absorbingsubstances, binding moieties such as biotin, mass tags, fluorescenttags, charge tags, volatile tags and hydrophobic tags. Additionalexamples of detection moieties that may be used in conjunction with thepresent invention are provided in U.S. Patent Publication No.20030194717 (application Ser. No. 10/221,666), which is herebyincorporated by reference.

The term “compomer” as used herein is a molecule synthesized in a targetdetection assay from a compomer template to indirectly indicate thepresence of a particular target molecule in a sample being assayed.Compomers are comprised of one or more subunits. Particularly preferredsubunits for compomer polymerization are nucleobase subunits. Compomersare described in greater detail in US Patent Application 20050287533(Ser. No. 10/874,898), which is hereby incorporated in its entirety byreference.

The term “nucleoside modification” as used herein refers to alterationsof the detector oligonucleotide at the molecular level (e.g., basemoiety, sugar moiety or phosphate backbone). Nucleoside modificationsinclude, but are not limited to, the introduction of cleavage blockersor cleavage inducers, the introduction of minor groove binders, isotopicenrichment, isotopic depletion, the introduction of deuterium, andhalogen modifications. Nucleoside modifications may also includemoieties that increase the stringency of hybridization or increase themelting temperature of the detector oligonucleotide. For example, anucleotide molecule may be modified with an extra bridge connecting the2′ and 4′ carbons resulting in locked nucleic acid (LNA) nucleotide thatis resistant to cleavage by a nuclease or may be abasic in nature toprevent non-specific or specific complementary binding as with1′,2′-Dideoxyribose.

The term “specific” or “specificity” in reference to the binding of onemolecule to another molecule, such as a probe for a targetpolynucleotide, refers to the recognition, contact, and formation of astable complex between the two molecules, together with substantiallyless recognition, contact, or complex formation of that molecule withother molecules. As used herein, the term “anneal” refers to theformation of a stable complex between two molecules.

A probe is “capable of hybridizing” to a nucleic acid sequence if atleast one region of the probe shares substantial sequence identity withat least one region of the complement of the nucleic acid sequence.“Substantial sequence identity” is a sequence identity of at least about80%, preferably at least about 85%, more preferably at least about 90%,95% or 99%, and most preferably 100%. For the purpose of determiningsequence identity of a DNA sequence and a RNA sequence, U and T oftenare considered the same nucleotide. For example, a probe comprising thesequence ATCAGC is capable of hybridizing to a target RNA sequencecomprising the sequence GCUGAU.

The term “cleavage agent” as used herein refers to any means that iscapable of cleaving a detector oligonucleotide to yieldmass-distinguishable products, including but not limited to enzymes. Formethods wherein amplification does not occur, the cleavage agent mayserve solely to cleave, degrade or otherwise release the detectoroligonucleotide or fragments thereof. The cleavage agent may be anenzyme. The cleavage agent may be natural, synthetic, unmodified ormodified.

For methods wherein amplification occurs, the cleavage agent ispreferably an enzyme that possess synthetic (or polymerization) activityand nuclease activity. Such an enzyme is often a nucleic acidamplification enzyme. An example of a nucleic acid amplification enzymeis a nucleic acid polymerase enzyme such as Thermus aquaticus (Taq) DNApolymerase (TaqMAN®) or E. coli DNA polymerase I. The enzyme may benaturally occurring, unmodified or modified.

The term “polymerase” refers to an enzyme that catalyzes polynucleotidesynthesis by addition of nucleotide units to a nucleotide chain usingDNA or RNA as a template. The term refers to either a complete enzyme ora catalytic domain.

B. INTRODUCTION

The present invention pertains in part to quantitative amplificationprocesses employing a hybridization probe to be detected by massspectrometry and a reaction comprising a single enzyme capable ofsynthesis and nuclease activities. In contrast, certain quantitativeamplification applications described elsewhere require (a) ahybridization probe containing a detectable label, for example afluorescent tag, that is detected by a method other than massspectrometry (see, e.g., U.S. Pat. No. 5,210,015), or (b) multiplecleavage agents employed to cleave a cleavage structure in anon-amplification based method (see, e.g., U.S. Pat. No. 5,719,028). Inembodiments described herein, a detector oligonucleotide is included inthe amplification reaction along with primers that amplify the template.Further, the detector oligonucleotide or fragments thereof are detectedby mass spectrometry allowing for enhanced levels of multiplexing. Insome embodiments, the detector oligonucleotide is modified and in otherembodiments the detector oligonucleotide is unmodified. Theamplification conditions, enzyme properties, detector oligonucleotideproperties, detector oligonucleotide binding properties andmass-distinguishable product detection methods are described in furtherdetail herein hereafter.

C. AMPLIFICATION CONDITIONS

In one embodiment, the methods described herein employ modifiedquantitative amplification methods that allow for accurate and sensitivenucleic acid analysis in a multiplexed manner. The polymerase chainreaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202to Mullis and Mullis et al., describe a method for increasing theconcentration of a segment of target sequence in a mixture of genomicDNA without cloning or purification. PCR can be used to directlyincrease the concentration of the target to an easily detectable level.This process for amplifying the target sequence involves introducing amolar excess of two oligonucleotide primers which are complementary totheir respective strands of the double-stranded target sequence to theDNA mixture containing the desired target sequence. The mixture isdenatured and then allowed to hybridize. Following hybridization, theprimers are extended with polymerase so as to form complementarystrands. The steps of denaturation, hybridization, and polymeraseextension can be repeated as often as needed, in order to obtainrelatively high concentrations of a segment of the desired targetsequence.

Methods of the present invention allow for the amplification reaction tobe performed simultaneously in single, closed reaction vessel, whereinthe amplification step may be repeated until a signal is detected.Byproducts of this synthesis are oligonucleotide fragments from thedetector oligonucleotide which consist of a mixture of mono-, di- andlarger nucleotide fragments. The detector oligonucleotide may bemodified to include detection features detectable by mass spectrometryor the oligonucleotide fragments may be modified to yieldmass-distinguishable products of a given molecular mass or size.Repeated cycles of denaturation, detector oligonucleotide and primerannealing, and primer extension and cleavage of the detectoroligonucleotide result in the exponential accumulation of the targetregion defined by the primers and the exponential generation ofmass-distinguishable products. Sufficient cycles are run to achieve adetectable species of MDP's.

The present invention offers many advantages including the ability tomore easily design and perform multiplex assays, especially compared toassays currently available. In multiplex assays, several target nucleicacids can be detected simultaneously. In a multiplex format, sets ofspecially designed detector oligonucleotides are used such that theresulting mass-distinguishable products have a unique mass that can bedifferentiated from each other. A multiplex experiment can be used todetect 2 or more target nucleic acids, 10 or more target nucleic acids,100 or more target nucleic acids, or 1,000 or more target nucleic acidsin the same assay. The number of detector oligonucleotides used in amultiplex assay is equal to or greater than the number of target nucleicacids to be detected. For example, when a multiplex experiment is usedto detect 10 target nucleic acids, 10 or more detector oligonucleotidesthat result in 10 or more mass-distinguishable products of unique massare used. The number of analytes that can be detected in a single assayis limited only by the number of mass-distinguishable products that canbe detected in a single assay. As described herein, mass spectrometrycan resolve small differences in mass allowing the use of a large numberof detector oligonucleotides in a single assay.

D. PRIMER PROPERTIES

Oligonucleotide primers and probes can be prepared using any suitablemethod, such as, for example, methods using phosphotriesters andphosphodiesters well known to those skilled in the art. In someembodiments, one or more detection moieties are included in the detectoroligonucleotide. The oligonucleotide can also be modified at the basemoiety, sugar moiety, or phosphate backbone, for example, with minorgroove binders or intercalating agents.

The primers for the amplification reactions are designed according toknown algorithms. The primers are designed to hybridize to sequencesthat flank the target nucleic acid. Typically, commercially available orcustom software will use algorithms to design primers such that theannealing temperatures are close to melting temperature. Amplificationprimers are usually at least 12 bases, more often about 15, 18, or 20bases in length. Primers are typically designed so that all primersparticipating in a particular reaction have melting temperatures thatare within 5 degree C., and most preferably within 2 degree C. of eachother. Primers are further designed to avoid priming on themselves oreach other. Primer concentration often is sufficient to bind to theamount of target sequences that are amplified so as to provide anaccurate assessment of the quantity of amplified sequence. Those ofskill in the art will recognize that the concentration of primer willvary according to the binding affinity of the primers as well as thequantity of sequence to be bound. Typical primer concentrations willrange from 0.01 μM to 1.0 μM. Also, the primer concentration may bealtered relative to the detector oligonucleotide concentration, whereinthe detector oligonucleotide concentration is greater than the primerconcentration.

In another embodiment, the forward and reverse primer concentrations maybe altered relative to each other (a condition sometimes referred to asAsymmetric PCR) to thereby to preferentially amplify one strand of thetemplate DNA more than the other. In a preferred embodiment, the strandon which the detector oligonucleotide binds is preferentially amplified.

The amplification reactions are incubated under conditions in which theprimers hybridize to the target sequence template and are extended by apolymerase. Such reaction conditions may vary, depending on the targetnucleic acid of interest and the composition of the primer. Theamplification reaction cycle conditions are selected so that the primershybridize specifically to the target template sequence and are extended.Primers that hybridize specifically to a target template amplify thetarget sequence preferentially in comparison to other nucleic acids thatmay be present in the sample that is analyzed.

E. ENZYME PROPERTIES

The present invention incorporates the use of a cleavage agent todegrade and release detectable mass-distinguishable products. Severalnucleases are known in the art that can be used to cleave differenttypes of nucleic acids. For example, nucleases are available that cancleave double-stranded DNA, for example, DNAse I and Exonuclease III, orsingle-stranded DNA, for example, nuclease S1. Nucleases include enzymesthat function solely as nucleases as well as multi-functional enzymesthat contain nuclease activity such as, for example, DNA polymeraseslike Taq polymerase that have 5′ nuclease activity. Several derivativesof Taq polymerases derived from different bacterial species or fromdesigned mutations are known which cleave specific structures of nucleicacid hybrids (Kaiser et al., J. Biol. Chem. 274:21387-21394 (1999);Lyamichev et al., Proc. Natl. Acad. Sci. USA 96:6143-6148 (1999); Ma etal., J. Biol. Chem. 275:24693-24700 (2000)). For example, Cleavaseenzymes (Third Wave Technologies) have been developed that cleave onlyat specific nucleic acid structures. In a preferred embodiment, thecleavage agent is an enzyme with polymerase and nuclease activity,wherein the target nucleic acid is exponentially amplified whilemass-distinguishable products are generated.

In some embodiments, the enzyme cleaves the detector oligonucleotide onenucleotide into the 5′ end of the detector oligonucleotide region thatis hybridized to the target. In another embodiment, the detectoroligonucleotide is not cleaved nucleotide-by-nucleotide. Instead,cleavage is regulated through the introduction of detection moieties ornucleoside modifications that yield mass-distinguishable products ofgreater than one nucleotide. In a preferred embodiment, a detectoroligonucleotide is cleaved in such a way that it yields reproducible anddistinguishable MDP's.

F. DETECTOR OLIGONUCLEOTIDE PROPERTIES

The detector oligonucleotides of the invention can be any suitable size,and are typically in the range of from about 6 to about 100 nucleotides,more preferably from about 6 to about 80 nucleotides and even morefrequently from about 10 to about 40 nucleotides. The precise sequenceand length of a detector oligonucleotide depends in part on the natureof the target polynucleotide to which it binds. The binding location andlength may be varied to achieve appropriate annealing and meltingproperties for a particular embodiment. Guidance for making such designchoices can be found in many art recognized references. Hybridization ofthe detector oligonucleotide, in conjunction with amplification of thetarget sequence with primers to amplify the target nucleic acid,provides a quantitative determination of the amount of the targetnucleic acid sequence in a sample. In a preferred embodiment, thedetector oligonucleotide is non-extendable (e.g., through theintroduction of a 3′ dideoxynucleotide), thereby reducing theprobability of amplification artifacts.

The detector oligonucleotide may also contain a mismatch to the targetnucleic acid sequence, e.g., at an invariant (nonpolymorphic) positionof the target nucleic acid sequence. In some embodiments, additionalnucleotides, e.g., two, three, four, five, six, or seven or morenucleotides, can also be mismatched to the target nucleic acid. In someembodiments, the additional mismatches form a stem-loop structure withupstream detector oligonucleotide sequences prior to hybridization withthe target nucleic acid sequence. The mismatch nucleotides may code fora nickase site. The mismatch nucleotides may be present at the 5′ end,3′ end or internal to the detector oligonucleotide. Examples of 3′mismatches are described in US Patent Application No. 20060024695, whichis hereby incorporated by reference. In another embodiment, the 5′ endof the complementary region of the detector oligonucleotide may containa GC clamp.

The detector oligonucleotides may be part of a library that are designedto yield a range of mass distinguishable products (or species) that canbe detected individually or as part of a multiplexed reaction. Thelibrary of detector oligonucleotides may designed, including theincorporation of modifications, to improve binding specificity to thetarget nucleic acid, to generate uniform cleavage products and/or tomodify the detection properties (e.g., mass) of the MDP's to improvemultiplexing capabilities. In one aspect, the library comprises two ormore detector oligonucleotide species, wherein each species comprises a3′ portion that is complementary or substantially complementary to thetarget nucleic acid and a 5′ portion that is susceptible to cleavage bya cleavage agent, and thereby produces a mass distinguishable product(MDP) that is unique to each detector oligonucleotide species and eachcorresponding target nucleic acid. In another embodiment, thecomplementary portion of a detector oligonucleotide species is modifiedto include one or more modifications such as minor groove bindingmoieties and locked nucleic acids (LNA's). In another embodiment, thenon-complementary 5′ portion of a detector oligonucleotide species ismodified to include one or more modifications such as abasicnucleotides, a capture mechanism (e.g., biotin or an oligonucleotidecomplementary to a capture probe (but not the target nucleic acid)), areleasing mechanism for subsequent release after capture (e.g.,photocleavable linker) and mass modifiers.

Detector oligonucleotides may be modified or unmodified. Modifiedoligonucleotides may contain detection moieties or nucleosidemodification. Examples of detection moieties and nucleosidemodifications, and methods of making and using them, are described inU.S. Pat. No. 5,174,962; U.S. Pat. No. 5,360,819; U.S. Pat. No.5,516,931; U.S. Pat. No. 6,268,129; U.S. Pat. No. 6,635,452; U.S. Pat.No. 6,322,980; U.S. Pat. No. 6,514,700; U.S. Pat. No. 6,649,351; andU.S. Pat. No. 6,613,509; and US Patent Application No. US 20060172319,all of which are hereby incorporated by reference.

The term “detection moiety” refers to a mass label, tag or signal.Examples of the types of detection moieties for the present inventioninclude a repertoire of compounds, preferably ones that share similarmass spectrometric desorption properties and have similar or identicalcoupling chemistries in order to streamline synthesis of multipledetection moiety variants. A detection moiety of the present inventionis detectable by mass spectrometry. Representative types of massspectrometric techniques include matrix-assisted laser desorptionionization, direct laser-desorption, electrospray ionization, secondaryneutral, and secondary ion mass spectrometry, with laser-desorptionionization being preferred. The dynamic range of mass spectralmeasurements can generally be extended by use of a logarithmic amplifierand/or variable attenuation in the processing and analysis of thesignal.

In other related embodiments, the nucleotides can be labeled with anytype of chemical group or moiety that allows for detection, cleavage,cleavage resistance or non-binding modification of the detectoroligonucleotide including but not limited to radioactive molecules,fluorescent molecules, antibodies, antibody fragments, haptens,carbohydrates, biotin, derivatives of biotin, phosphorescent moieties,luminescent moieties, electrochemiluminescent moieties, chromaticmoieties, and moieties having a detectable electron spin resonance,electrical capacitance, dielectric constant or electrical conductivity.The nucleotides can be labeled with one or more than one type ofchemical group or moiety. Each nucleotide can be labeled with the samechemical group or moiety. Alternatively, each different nucleotide canbe labeled with a different chemical group or moiety. The labelednucleotides can be dNTPs, ddNTPs, or a mixture of both dNTPs and ddNTPs.The unlabeled nucleotides can be dNTPs, ddNTPs or a mixture of bothdNTPs and ddNTPs.

Any combination of nucleotides can be used to incorporate nucleotidesincluding but not limited to unlabeled deoxynucleotides, labeleddeoxynucleotides, unlabeled dideoxynucleotides, labeleddideoxynucleotides, a mixture of labeled and unlabeled deoxynucleotides,a mixture of labeled and unlabeled dideoxynucleotides, a mixture oflabeled deoxynucleotides and labeled dideoxynucleotides, a mixture oflabeled deoxynucleotides and unlabeled dideoxynucleotides, a mixture ofunlabeled deoxynucleotides and unlabeled dideoxynucleotides, a mixtureof unlabeled deoxynucleotides and labeled dideoxynucleotides,dideoxynucleotide analogues, deoxynucleotide analogues, a mixture ofdideoxynucleotide analogues and deoxynucleotide analogues,phosphorylated nucleoside analogues, 2′-deoxynucleotide-5′-triphosphate,and modified 2′-deoxynucleotide-5′-triphosphate.

All four nucleotides can be labeled with different fluorescent groups,which will allow one reaction to be performed in the presence of allfour labeled nucleotides. Alternatively, four separate “fill in”reactions can be performed for each locus of interest; each of the fourreactions will contain a different labeled nucleotide (e.g. ddATP*,ddTTP*, ddGTP*, or ddCTP*, where * indicates a labeled nucleotide). Eachnucleotide can be labeled with different chemical groups or the samechemical groups. The labeled nucleotides can be dideoxynucleotides ordeoxynucleotides.

In another embodiment, nucleotides can be labeled with fluorescent dyesincluding but not limited to fluorescein, pyrene, 7-methoxycoumarin,Cascade Blue™, Alexa Flur 350, Alexa Flur 430, Alexa Flur 488, AlexaFlur 532, Alexa Flur 546, Alexa Flur 568, Alexa Flur 594, Alexa Flur633, Alexa Flur 647, Alexa Flur 660, Alexa Flur 680, AMCA-X,dialkylaminocoumarin, Pacific Blue, Marina Blue, BODIPY 493/503, BODIPYFI-X, DTAF, Oregon Green 500, Dansyl-X, 6-FAM, Oregon Green 488, OregonGreen 514, Rhodamine Green-X, Rhodol Green, Calcein, Eosin, ethidiumbromide, NBD, TET, 2′, 4′, 5′, 7′ tetrabromosulfonefluorescien,BODIPY-R6G, BODIPY-FI BR2, BODIPY 530/550, HEX, BODIPY 558/568,BODIPY-TMR-X, PyMPO, BODIPY 564/570, TAMRA, BODIPY 576/589, Cy3,Rhodamine Red-x, BODIPY 581/591, carboxyrhodamine, Texas Red-X,BODIPY-TR-X, Cy5, SpectrumAqua, SpectrumGreen #1, SpectrumGreen #2,SpectrumOrange, SpectrumRed, or naphthofluorescein.

Detection moieties may include a vast array of different types ofcompounds including biopolymers and synthetic polymers. Representativebiological monomer units that may be used as detection moieties, eithersingly or in polymeric form, include peptide nucleic acids (PNAs),nucleotide-peptide conjugates, amino acids, nonnatural amino acids,nucleic acids, saccharides, carbohydrates, peptide mimics and nucleicacid mimics. Preferred peptides are naturally occurring, stable andrelatively small (e.g., the neuropeptide Substance P or c-myc).Preferred amino acids also include those with simple aliphatic sidechains (e.g., glycine, alanine, valine, leucine and isoleucine), aminoacids with aromatic side chains (e.g., phenylalanine, tryptophan,tyrosine, and histidine), amino acids with oxygen and sulfur containingside chains (e.g., serine, threonine, methionine and cysteine), aminoacids with side chains containing carboxylic or amide groups (e.g.,aspartic acid, glutamic acid, asparagine and glutamine), and amino acidswith side chains containing strongly basic groups (e.g., lysine andarginine), and proline. Derivatives of the above described amino acidsare also contemplated as monomer units. An amino acid derivative as usedherein is any compound that contains within its structure the basicamino acid core of an a amino-substituted carboxylic acid, withrepresentative examples including but not limited to azaserine,fluoroalanine, GABA, ornithine, norleucine and cycloserine. Peptidesderived from the above described amino acids can also be used as monomerunits. Representative examples include both naturally occurring andsynthetic peptides with molecular weight above about 500 Daltons, withpeptides from about 500-5000 Daltons being preferred. Representativeexamples of saccharides include ribose, arabinose, xylose, glucose,galactose and other sugar derivatives composed of chains from 2-7carbons. Representative polysaccharides include combinations of thesaccharide units listed above linked via a glycosidic bond. Generally,the sequence of the polymeric units within any one detection moiety isnot critical; the total mass is the key feature of the label. In anembodiment of the invention, peptide detection moieties are combinedwith nucleotides to yield a library of MDP's. For example, the samepeptide (e.g., Substance P: an 11-amino acid polypeptide with thesequence: Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met (SEQ ID NO: 1)) isconjugated to a library of nucleotides of differing masses (e.g., allpossible 4-mers).

The monomer units according to the present invention also may becomposed of nucleobase compounds or nucleoside modifications. As usedherein, the term nucleobase refers to any moiety that includes withinits structure a purine, a pyrimidine, a nucleic acid, nucleoside, abasicnucleotide, nucleotide or derivative of any of these, such as aprotected nucleobase, purine analog, pyrimidine analog, folinic acidanalog, methyl phosphonate derivatives, phosphotriester derivatives,borano phosphate derivatives or phosphorothioate derivatives.

Detection moieties according to the present invention may also includeany organic or inorganic polymer that has a defined mass value, remainswater soluble during bioassays and is detectable by mass spectrometry.Representative synthetic monomer units that may be used as mass units inpolymeric form include polyethylene glycols, polyvinyl phenols,polymethyl methacrylates, polypropylene glycol, polypyroles, andderivatives thereof. A wide variety of polymers would be readilyavailable to one of skill in the art. The polymers may be composed of asingle type of monomer unit or combinations of monomer units to create amixed polymer. The sequence of the polymeric units within any onedetection moiety is not critical; the total mass is the key feature ofthe label.

For nonvolatile detection moieties having mass below about 500 Da,usually significant ionic character is required; representative examplesinclude polyethylene glycol oligomers of quaternary ammonium salts(e.g., R—(O—CH.sub.2-CH.sub.2).sub.n-N(CH.sub.3).sub.3.sup.+.Cl.sup.−)and polyethylene glycol oligomers of carboxylic acids and salts (e.g.,R—(O—CH.sub.2-CH.sub.2).sub.n-CO.sub.2-.Na.sup.+).

Examples of involatile detection moieties typically include smalloligomers of polyethylene glycol and small peptides (natural ormodified) less than about 500 Da in molecular weight. In theseinstances, as for all of the cases considered herein, mass analysis isnot by electron attachment.

Detection moieties of the present invention may also include a varietyof nonvolatile and involatile organic compounds which are nonpolymeric.Representative examples of nonvolatile organic compounds include hemegroups, dyes, organometallic compounds, steroids, fullerenes, retinoids,carotenoids and polyaromatic hydrocarbons.

It is preferable when using multiple detection moieties on a detectoroligonucleotide, to avoid signal overlap. In addition to presenting alarge, primary signal for a detection moiety with a single charge, thereis also the potential for multiply charged versions of a detectionmoiety to present a signal as well as dimerized versions of a detectionmoiety. The presence of multiple signals for a single detection moietycan potentially overlap with and obscure the signal for the primary peakof a second detection moiety. Thus typically the range of detectionmoieties used for a given analysis may have a mass range where nomultiply charged or dimer species can interfere with the detection ofall detection moieties, for example, the detection moieties may have arange of masses wherein the smallest mass-label is more than half themass of the largest detection moiety.

Other detection moieties include base-linked fluors and quenchers, whichare well-known in the art. They can be obtained, for example, from LifeTechnologies (Gaithersburg, Md.), Sigma-Genosys (The Woodlands, Tex.),Invitrogen (Carlsbad, Calif.), or Synthetic Genetics (San Diego,Calif.). In some cases, base-linked fluors are incorporated into theoligonucleotides by post-synthesis modification of oligonucleotides thatwere synthesized with reactive groups linked to bases. The fluor can beattached to the 3′ OH of the sugar or the base. Base-linked fluorsand/or quenchers may be used to create unique MDP's of a particularmass, which can then be detected by mass spectrometry.

In another embodiment, the detector oligonucleotide comprisesnon-cleavable (i.e., non-degradable or nuclease-resistant) nucleotides.As described herein, an enzyme of the invention can cleave any bonds inthe detector oligonucleotide that are nuclease-susceptible. However, anadvantage of having at least one nuclease-resistant bond in thetarget-binding moiety (i.e., the portion of the detector oligonucleotidethat hybridizes with or is complementary to the target nucleic acid) isthat a detector oligonucleotide will yield a single-sized species ofmass-distinguishable product upon cleavage. Examples of particularlypreferred non-cleavable (or cleavage resistant) nucleotides includeamadites such as 2′-O-Methyl, 2′-Fluoro and Phosphothiote, for example.Another example of a non-cleavable nucleotide is a locked nucleic acid(LNA). Locked nucleic acid, also referred to as inaccessible RNA, is amodified RNA nucleotide. The ribose moiety of an LNA nucleotide is oftenmodified with an extra bridge connecting the 2′ and 4′ carbons. LNAnucleotides can be incorporated in with DNA or RNA bases in theoligonucleotide detector whenever desired. In one embodiment, the one ormore LNAs are incorporated into the 5′ end of the complementary (ortarget-binding moiety) region of the detector oligonucleotide.

The locked ribose conformation enhances base stacking and backbonepre-organization, and thereby significantly increases the thermalstability (melting temperature) of the detector oligonucleotides wherethe LNA's are incorporated. This effect may be further enhanced byplacing two or more LNAs adjacent to each other. See Example 3 below.

Nuclease-cleavable bonds can include, for example, a phosphodiesterbond, and nuclease-resistant bonds can include, for example,thiophosphate, phosphinate, methylphosphonate, phosphoramidate, or alinker other than a phosphorous acid derivative, such as amide andboronate linkages or alkylsilyldiester and peptide nucleic acid.

In another embodiment, the detector oligonucleotide comprises groups orlinkages cleavable by an enzyme. Enzymatically-cleavable release groupsinclude phosphodiester or amide linkages as well as restrictionendonuclease recognition sites.

In another embodiment, the detector oligonucleotide comprises a minorgroove binder (MGB), wherein the MGB is on the 5′ end, middle, or 3′ endof the detector oligonucleotide—depending on the particular assay. Minorgroove binding proteins and/or a modified base DNA probes withconjugated minor groove binder (MGB) groups form extremely stableduplexes with single-stranded DNA targets, allowing shorter probes to beused for hybridization based assays (e.g., U.S. Pat. No. 5,801,155).Accordingly, in some embodiments, minor groove binder groups are alsoincluded in the detector oligonucleotide, e.g., at the 3′ end of theprobe. A variety of suitable minor groove binders have been described inthe literature. See, for example, U.S. Pat. No. 5,801,155; Wemmer &Dervan, Current Opinion in Structural Biology 7:355-361-(1997); Walker,et al., Biopolymers 44:323-334 (1997); Zimmer & Wahnert, Prog. Biophys.Molec. Bio. 47:31-112 (1986); and Reddy, et al., Pharmacol. Therap.84:1-111 (1999). Suitable methods for attaching MGBs (as well as othermoieties) through linkers to oligonucleotides are described in, forexample, U.S. Pat. Nos. 5,512,677; 5,419,966; 5,696,251; 5,585,481;5,942,610 and 5,736,626, all of which are hereby incorporated byreference.

In another embodiment, the detector oligonucleotide comprisesisotopically-coded nucleotides. In one example, an allele-specificdetector oligonucleotide comprises an isotopically-coded nucleotide thathybridizes to the SNP allele. The isotopically-coded detectoroligonucleotide in turn yields an isotopically-codedmass-distinguishable product. In one embodiment, only singleisotopically-coded nucleotides or short fragments comprisingisotopically-coded nucleotides (e.g., two, three or four bases) aregenerated and detected by mass spectrometry. When only singlenucleotides are detected, purification is simplified since phosphatebackbones are not present and salt adduct formation is minimized. See,for example U.S. Pat. No. 6,613,509 (hereby incorporated by reference),which describes incorporation of isotopes into nucleic acids.

In another embodiment, the detector oligonucleotide may be modified toincrease its melting temperature (Tm). In one embodiment, the primermelting temperature often is around 58-60° C., and detectoroligonucleotide Tm often is 10° C. higher than the primer's Tm. The Tmof both the primers often is approximately equal.

G. DETECTOR OLIGONUCLEOTIDE BINDING PROPERTIES

In some embodiments, degradation of the detector oligonucleotide isperformed under conditions wherein one or more of the nucleic acids inthe structure can disassociate from the target. In one embodiment, fullor partial disassociation of the detector oligonucleotide allows theformation of multiple mass-distinguishable products. In someembodiments, said disassociation is induced by an increase intemperature, such that one or more oligonucleotides can no longerhybridize to the target strand. In other embodiments, saiddisassociation occurs because cleavage of an oligonucleotide producesonly cleavage products that cannot bind to the target strand under theconditions of the reaction. In a preferred embodiment, conditions areselected wherein an oligonucleotide may associate with (i.e., hybridizeto) and disassociate from a target strand regardless of cleavage. In aparticularly preferred embodiment, conditions are selected such that thenumber of copies of the detector oligonucleotide that can be cleavedwhen part of a duplex structure exceeds the number of copies of thetarget nucleic acid strand by a sufficient amount that when thepartially cleaved detector oligonucleotide disassociates, theprobability that the target strand will associate with an intact copy ofthe detector oligonucleotide is greater than the probability that itwill associate with a cleaved copy of the detector oligonucleotide.

H. MASS-DISTINGUISHABLE PRODUCT DETECTION METHODS

Mass-distinguishable products are distinguished by a particular physicalattribute or detection feature, including but not limited to length,mass, charge, or charge-to-mass ratio. In a preferred embodiment, thedetection feature is mass. In another related embodiment, the MDP may bedistinguished by a behavior that is related to a physical attribute,including but not limited to mass, time of flight in MALDI-TOF massspectrometry. In a related embodiment, MDP's from one or more detectoroligonucleotides are released and selectively desorbed from a massspectral matrix such that the non-selective primers and detectoroligonucleotides (i.e., the target nucleic acid is not present) do notdesorb. For these embodiments, the MDP's should desorb more efficientlyfrom the mass spectral matrix than detector oligonucleotides or othernon-MDP's present in the reaction mixture. Preferred mass spectralmatrices include 2,5-dihydroxybenzoic acid,alpha-cyano-4-hydroxycinammic acid, 3-hydroxypicolinic acid (3-HPA),di-ammoniumcitrate (DAC) and combinations thereof. In anotherembodiment, the mass spectral matrices may be designed for the analysisof proteins. Exemplary matrices for protein analysis include, but arenot limited to, DHB and CHCA.

The method can further include an additional step of separating one ormore detector oligonucleotide fragments (i.e., MDP's) from un-cleaved orpartially-cleaved detector oligonucleotides. Separation can beaccomplished using capture ligands, such as biotin or other affinityligands, and capture agents, such as avidin, streptavidin, an antibody,a receptor, a capture probe that is complementary to the MDP, or afunctional fragment thereof, having specific binding activity to thecapture ligand. A MDP can contain a capture ligand having specificbinding activity for a capture agent. For example, the MDP can bebiotinylated or attached to an affinity ligand using methods well knownin the art. See Example 4 below. A capture ligand and capture agent canalso be used to add mass to the remaining part of the MDP such that itcan be excluded from the mass range of the MDP detected in a massspectrometer. In one embodiment, the capture probe may have a universalprimer for universal amplification of cleavage product. In anotherembodiment the captured MDP may be released from its affinity moleculeusing any method known in the art. Modes of releasing MDP's include, butare not limited to, enzymatic cleavage, photocleavage, exchangereactions, temperature-sensitive release, and release based on chemicalproperties of the MDP molecule.

A separation step can also be used to remove salts, enzymes, or otherbuffer components from the MDP's. Several methods well known in the art,such as chromatography, gel electrophoresis, or precipitation, can beused to clean up the sample. For example, size exclusion chromatographyor affinity chromatography can be used to remove salt from a sample. Thechoice of separation method can depend on the amount of a sample. Forexample, when small amounts of sample are available or a miniaturizedapparatus is used, a micro-affinity chromatography separation step canbe used. In addition, whether a separation step is desired, and thechoice of separation method, can depend on the detection method used.For example, the efficiency of matrix-assisted laserdesorption/ionization and electrospray ionization can be improved byremoving salts from a sample. For example, salts can absorb energy fromthe laser in matrix-assisted laser desorption/ionization and result inlower ionization efficiency.

Mass spectrometry is the preferred method to detect mass-distinguishableproducts of the invention and thus identify and/or quantitate targetnucleic acids. Mass-distinguishable products can be ionized in a massspectrometer and the ions separated in space or time based on theirmass-to-charge ratio. The mass spectrometer then calculates a massassociated with each ion. Therefore, when referring to massspectrometry, the term mass can be used for simplicity to describe amass-to-charge ratio.

Mass spectrometry is a sensitive and accurate technique for separatingand identifying molecules. Generally, mass spectrometers have two maincomponents, an ion source for the production of ions and amass-selective analyzer for measuring the mass-to-charge ratio of ions,which is and converted into a measurement of mass for these ions.Several ionization methods are known in the art and described herein. Amass-distinguishable product can be charged prior to, during or aftercleavage from the detector oligonucleotide. Consequently, amass-distinguishable product that will be measured by mass spectrometrydoes not always require a charge since a charge can be acquired throughthe mass spectrometry procedure. In mass spectrometry analysis, optionalcomponents of a MDP such as charge and detection moieties can be used tocontribute mass to the MDP.

Different mass spectrometry methods, for example, quadrupole massspectrometry, ion trap mass spectrometry, time-of-flight massspectrometry, gas chromatography mass spectrometry and tandem massspectrometry, as described herein, can utilize various combinations ofion sources and mass analyzers which allows for flexibility in designingcustomized detection protocols. In addition, mass spectrometers can beprogrammed to transmit all ions from the ion source into the massspectrometer either sequentially or at the same time. Furthermore, amass spectrometer can be programmed to select ions of a particular massfor transmission into the mass spectrometer while blocking other ions.

The ability to precisely control the movement of ions in a massspectrometer allows for greater options in detection protocols which canbe advantageous when a large number of mass-distinguishable products,for example, from a multiplex experiment, are being analyzed. Forexample, in a multiplex experiment with a large number of MDP's it canbe advantageous to select individual reporters from a group of similarreporters and then analyze that reporter separately. Another advantagebased on controlling the mass range detected by the mass spectrometerincludes the ability to exclude un-cleaved or partially-cleaved taggedprobes from being analyzed which reduces background noise from theassay.

Mass spectrometers can resolve ions with small mass differences andmeasure the mass of ions with a high degree of accuracy. Therefore,MDP's of similar masses can be used together in the same experimentsince the mass spectrometer can differentiate the mass of even closelyrelated tags. The high degree of resolution and mass accuracy achievedusing mass spectrometry methods allows the use of large sets of taggedprobes because the resulting reporter tags can be distinguished fromeach other. The ability to use large sets of tagged probes is anadvantage when designing multiplex experiments.

Another advantage of using mass spectrometry for detecting the mass of amass-distinguishable product is based on the high sensitivity of thistype of mass analysis. Mass spectrometers achieve high sensitivity byutilizing a large portion of the ions that are formed by the ion sourceand efficiently transmitting these ions through the mass analyzer to thedetector. Because of this high level of sensitivity, even limitedamounts of sample can be measured using mass spectrometry. This can bean advantage in a multiplex experiment where the amount of each MDPspecies may be small.

Mass spectrometry methods are well known in the art (see Burlingame etal. Anal. Chem. 70:647R-716R (1998); Kinter and Sherman, ProteinSequencing and Identification Using Tandem Mass SpectrometryWiley-Interscience, New York (2000)). The basic processes associatedwith a mass spectrometry method are the generation of gas-phase ionsderived from the sample, and the measurement of their mass.

The movement of gas-phase ions can be precisely controlled usingelectromagnetic fields generated in the mass spectrometer. The movementof ions in these electromagnetic fields is proportional to the m/z ofthe ion and this forms the basis of measuring the m/z and therefore themass of a sample. The movement of ions in these electromagnetic fieldsallows the ions to be contained and focused which accounts for the highsensitivity of mass spectrometry. During the course of m/z measurement,ions are transmitted with high efficiency to particle detectors thatrecord the arrival of these ions. The quantity of ions at each m/z isdemonstrated by peaks on a graph where the x axis is m/z and the y axisis relative abundance. Different mass spectrometers have differentlevels of resolution, that is, the ability to resolve peaks between ionsclosely related in mass. The resolution is defined as R=m/delta m, wherem is the ion mass and delta m is the difference in mass between twopeaks in a mass spectrum. For example, a mass spectrometer with aresolution of 1000 can resolve an ion with a m/z of 100.0 from an ionwith a m/z of 100.1.

Several types of mass spectrometers are available or can be producedwith various configurations. In general, a mass spectrometer has thefollowing major components: a sample inlet, an ion source, a massanalyzer, a detector, a vacuum system, and instrument-control system,and a data system. Difference in the sample inlet, ion source, and massanalyzer generally define the type of instrument and its capabilities.For example, an inlet can be a capillary-column liquid chromatographysource or can be a direct probe or stage such as used in matrix-assistedlaser desorption. Common ion sources are, for example, electrospray,including nanospray and microspray or matrix-assisted laser desorption.Exemplary mass analyzers include a quadrupole mass filter, ion trap massanalyzer and time-of-flight mass analyzer.

The ion formation process is a starting point for mass spectrumanalysis. Several ionization methods are available and the choice ofionization method depends on the sample to be analyzed. For example, forthe analysis of polypeptides a relatively gentle ionization proceduresuch as electrospray ionization (ESI) can be desirable. For ESI, asolution containing the sample is passed through a fine needle at highpotential which creates a strong electrical field resulting in a finespray of highly charged droplets that is directed into the massspectrometer. Other ionization procedures include, for example,fast-atom bombardment (FAB) which uses a high-energy beam of neutralatoms to strike a solid sample causing desorption and ionization.Matrix-assisted laser desorption ionization (MALDI) is a method in whicha laser pulse is used to strike a sample that has been crystallized inan UV-absorbing compound matrix. Other ionization procedures known inthe art include, for example, plasma and glow discharge, plasmadesorption ionization, resonance ionization, and secondary ionization. Amass-distinguishable product can become ionized prior to, during, orafter cleavage from the tagged probe.

Electrospray ionization (ESI) has several properties that are useful forthe invention described herein. For example, ESI can be used forbiological molecules such as polypeptides that are difficult to ionizeor vaporize. In addition, the efficiency of ESI can be very high whichprovides the basis for highly sensitive measurements. Furthermore, ESIproduces charged molecules from solution, which is convenient foranalyzing mass-distinguishable products that are in solution. Incontrast, ionization procedures such as MALDI require crystallization ofthe sample prior to ionization.

Since ESI can produce charged molecules directly from solution, it iscompatible with samples from liquid chromatography systems. For example,a mass spectrometer can have an inlet for a liquid chromatographysystem, such as an HPLC, so that fractions flow from the chromatographycolumn into the mass spectrometer. This in-line arrangement of a liquidchromatography system and mass spectrometer is sometimes referred to asLC-MS. A LC-MS system can be used, for example, to separate un-cleavedor partially cleaved MDP's from cleaved MDP's before mass spectrometryanalysis. In addition, chromatography can be used to remove salts orother buffer components from the MDP sample before mass spectrometryanalysis. For example, desalting of a sample using a reversed-phase HPLCcolumn, in-line or off-line, can be used to increase the efficiency ofthe ionization process and thus improve sensitivity of detection by massspectrometry.

A variety of mass analyzers are available that can be paired withdifferent ion sources. Different mass analyzers have differentadvantages as known to one skilled in the art and as described herein.The mass spectrometer and methods chosen for detection depends on theparticular assay, for example, a more sensitive mass analyzer can beused when a small amount of ions are generated for detection. Severaltypes of mass analyzers and mass spectrometry methods are describedbelow.

Ion mobility mass (IM) spectrometry is a gas-phase separation methodthat adds new dimensions to mass spectrometry (MS). IM separatesgas-phase ions based on their collision cross-section and can be coupledwith time-of-flight (TOF) mass spectrometry to yield a powerful toolused in the identification and characterization of proteins andpeptides. Therefore, IM-MS has particular utility for the presentinvention when the mass-distinguishable product is a protein or peptide.IM-MS is discussed in more detail by Verbeck et al. in the Journal ofBiomolecular Techniques (Vol 13, Issue 2, 56-61).

Quadrupole mass spectrometry utilizes a quadrupole mass filter oranalyzer. This type of mass analyzer is composed of four rods arrangedas two sets of two electrically connected rods. A combination of rf anddc voltages are applied to each pair of rods which produces fields thatcause an oscillating movement of the ions as they move from thebeginning of the mass filter to the end. The result of these fields isthe production of a high-pass mass filter in one pair of rods and alow-pass filter in the other pair of rods. Overlap between the high-passand low-pass filter leaves a defined m/z that can pass both filters andtraverse the length of the quadrupole. This m/z is selected and remainsstable in the quadrupole mass filter while all other m/z have unstabletrajectories and do not remain in the mass filter. A mass spectrumresults by ramping the applied fields such that an increasing m/z isselected to pass through the mass filter and reach the detector. Inaddition, quadruples can also be set up to contain and transmit ions ofall m/z by applying a rf-only field. This allows quadruples to functionas a lens or focusing system in regions of the mass spectrometer whereion transmission is needed without mass filtering. This will be of usein tandem mass spectrometry as described further below.

A quadrupole mass analyzer, as well as the other mass analyzersdescribed herein, can be programmed to analyze a defined m/z or massrange. This property of mass spectrometers is useful for the inventiondescribed herein. Since the mass range of cleaved mass-distinguishableproducts will be known prior to an assay, a mass spectrometer can beprogrammed to transmit ions of the projected correct mass range whileexcluding ions of a higher or lower mass range. The ability to select amass range can decrease the background noise in the assay and thusincrease the signal-to-noise ratio. In addition, a defined mass rangecan be used to exclude analysis of any un-cleaved detectoroligonucleotides, which would be of higher mass than the mass of themass-distinguishable products. Therefore, the mass spectrometer canaccomplish an inherent separation step as well as detection andidentification of the mass-distinguishable products.

Ion trap mass spectrometry utilizes an ion trap mass analyzer. In thesemass analyzers, fields are applied so that ions of all m/z are initiallytrapped and oscillate in the mass analyzer. Ions enter the ion trap fromthe ion source through a focusing device such as an octapole lenssystem. Ion trapping takes place in the trapping region beforeexcitation and ejection through an electrode to the detector. Massanalysis is accomplished by sequentially applying voltages that increasethe amplitude of the oscillations in a way that ejects ions ofincreasing m/z out of the trap and into the detector. In contrast toquadrupole mass spectrometry, all ions are retained in the fields of themass analyzer except those with the selected m/z. One advantage to iontraps is that they have very high sensitivity, as long as one is carefulto limit the number of ions being tapped at one time. Control of thenumber of ions can be accomplished by varying the time over which ionsare injected into the trap. The mass resolution of ion traps is similarto that of quadrupole mass filters, although ion traps do have low m/zlimitations.

Time-of-flight mass spectrometry utilizes a time-of-flight massanalyzer. For this method of m/z analysis, an ion is first given a fixedamount of kinetic energy by acceleration in an electric field (generatedby high voltage). Following acceleration, the ion enters a field-free or“drift” region where it travels at a velocity that is inverselyproportional to its m/z. Therefore, ions with low m/z travel morerapidly than ions with high m/z. The time required for ions to travelthe length of the field-free region is measured and used to calculatethe m/z of the ion.

One consideration in this type of mass analysis is that the set of ionsbeing studied be introduced into the analyzer at the same time. Forexample, this type of mass analysis is well suited to ionizationtechniques like MALDI which produces ions in short well-defined pulses.Another consideration is to control velocity spread produced by ionsthat have variations in their amounts of kinetic energy. The use oflonger flight tubes, ion reflectors, or higher accelerating voltages canhelp minimize the effects of velocity spread. Time-of-flight massanalyzers have a high level of sensitivity and a wider m/z range thanquadrupole or ion trap mass analyzers. Also data can be acquired quicklywith this type of mass analyzer because no scanning of the mass analyzeris necessary.

Gas chromatography mass spectrometry offers a nice solution fordetecting a target in real-time. The gas chromatography (GC) portion ofthe system separates the chemical mixture into pulses of analyte (e.g.,MDP's) and the mass spectrometer (MS) identifies and quantifies theanalyte.

Tandem mass spectrometry can utilize combinations of the mass analyzersdescribed above. Tandem mass spectrometers can use a first mass analyzerto separate ions according to their m/z in order to isolate an ion ofinterest for further analysis. The isolated ion of interest is thenbroken into fragment ions (called collisionally activated dissociationor collisionally induced dissociation) and the fragment ions areanalyzed by the second mass analyzer. These types of tandem massspectrometer systems are called tandem in space systems because the twomass analyzers are separated in space, usually by a collision cell.Tandem mass spectrometer systems also include tandem in time systemswhere one mass analyzer is used, however the mass analyzer is usedsequentially to isolate an ion, induce fragmentation, and then performmass analysis.

Mass spectrometers in the tandem in space category have more than onemass analyzer. For example, a tandem quadrupole mass spectrometer systemcan have a first quadrupole mass filter, followed by a collision cell,followed by a second quadrupole mass filter and then the detector.Another arrangement is to use a quadrupole mass filter for the firstmass analyzer and a time-of-flight mass analyzer for the second massanalyzer with a collision cell separating the two mass analyzers. Othertandem systems are known in the art including reflectron-time-of-flight,tandem sector and sector-quadrupole mass spectrometry.

Mass spectrometers in the tandem in time category have one mass analyzerthat performs different functions at different times. For example, anion trap mass spectrometer can be used to trap ions of all m/z. A seriesof rf scan functions are applied which ejects ions of all m/z from thetrap except the m/z of ions of interest. After the m/z of interest hasbeen isolated, an rf pulse is applied to produce collisions with gasmolecules in the trap to induce fragmentation of the ions. Then the m/zvalues of the fragmented ions are measured by the mass analyzer. Ioncyclotron resonance instruments, also known as Fourier transform massspectrometers, are an example of tandem-in-time systems.

Several types of tandem mass spectrometry experiments can be performedby controlling the ions that are selected in each stage of theexperiment. The different types of experiments utilize different modesof operation, sometimes called “scans,” of the mass analyzers. In afirst example, called a mass spectrum scan, the first mass analyzer andthe collision cell transmit all ions for mass analysis into the secondmass analyzer. In a second example, called a product ion scan, the ionsof interest are mass-selected in the first mass analyzer and thenfragmented in the collision cell. The ions formed are then mass analyzedby scanning the second mass analyzer. In a third example, called aprecursor ion scan, the first mass analyzer is scanned to sequentiallytransmit the mass analyzed ions into the collision cell forfragmentation. The second mass analyzer mass-selects the product ion ofinterest for transmission to the detector. Therefore, the detectorsignal is the result of all precursor ions that can be fragmented into acommon product ion. Other experimental formats include neutral lossscans where a constant mass difference is accounted for in the massscans. The use of these different tandem mass spectrometry scanprocedures can be advantageous when large sets of reporter tags aremeasured in a single experiment as with multiplex experiments.

In typical applications, the amount of mass-distinguishable productgenerated by the during the reaction is determined based on cyclethreshold (Ct) value, which represents the number of cycles required togenerate a detectable amount of nucleic acid. Determination of Ct valuesis well known in the art. Briefly, during PCR, as the amount of formedamplicon increases, the signal intensity increases to a measurable leveland reaches a plateau in later cycles when the reaction enters into anon-logarithmic phase. By plotting signal intensity versus the cyclenumber during the logarithmic phase of the reaction, the specific cycleat which a measurable signal is obtained can be deduced and used tocalculate the quantity of the target before the start of the PCR.Exemplary methods of determining Ct are described in, e.g., Heid et al.Genome Methods 6:986-94, 1996, with reference to hydrolysis probes.

For quantification, one may choose to use controls, which provide asignal in relation to the amount of the target that is present or isintroduced. A control to allow conversion of relative mass signals intoabsolute quantities is accomplished by addition of a known quantity of amass tag or mass label to each sample before detection of themass-distinguishable products. See for example, Ding and Cantor ProcNatl Acad Sci USA. 2003 Mar. 18; 100(6):3059-64, who describe a methodfor quantitative gene expression analysis, wherein the controlnucleotide contains an artificial single nucleotide polymorphism todistinguish it from the gene of interest. Any mass tag that does notinterfere with detection of the MDP's can be used for normalizing themass signal. Such standards preferably have separation properties thatare different from those of any of the molecular tags in the sample, andcould have the same or a different mass signatures.

I. NON-MASS-RELATED DETECTION METHODS

In another aspect, the present invention includes non-massspectrometry-based methods for detecting MDP's—both directly andindirectly. Examples of other, non-mass-related methods for detectingand/or quantifying MDP's include the use of hybridization probes,quantitative real time polymerase chain reaction (QRT-PCR), singlemolecule sequencing (e.g., digital PCR), electrophoresis,pyrosequencing, primer extension reactions, microarrays, nanoporesequencing, the use of chips and/or capture substrates and sequencing bysynthesis. In one embodiment, an MDP may have one or moredistinguishable properties (e.g., a unique mass and one or morefluorescent tags) that are detectable by multiple detection methods suchas mass spectrometry and fluorescence detection. For example, an MDP mayhave a series of labels (e.g., fluorescent tags or hybridized probes)that are detectable (e.g., optically or voltage differential) as the MDPpasses by or through a nanopore, enzyme or any other means for“threading” the MDP along a substrate. Additional sequencing methods aredescribed by Blow (Nature Methods 5(3): 267-274 (2008)) and Lin et al(Recent Patents in Biomedical Engineering 1(1): 60-67 (2008)), both ofwhich are hereby incorporated by reference.

J. COMPOSITIONS AND KITS

In another aspect, the present invention includes kits for performingthe methods of the invention, such kits comprising a primers (e.g.,universal primers) and detector oligonucleotides for detecting ormeasuring one or more target nucleic acids. Such kits further comprisingan enzyme and appropriate buffers for performing amplification reactionsthat cleave and release detector oligonucleotides or fragments thereoffor detection. In certain embodiments, a kit may include one or moredetector oligonucleotides that can result in one or moremass-distinguishable products, and one or more reagents associated withmass spectrometry, the latter of which may be, for example, one or moremass standards (e.g., for use as an internal standard), a matrix formatrix-assisted laser desorption ionization (MALDI) mass spectrometry(e.g., 3-hydroxypicolinic acid), a nucleic acid binding resin (e.g., C¹⁸resin), and/or a solution for conditioning a nucleic acid (e.g., a saltsolution).

EXAMPLES

The examples provided hereafter illustrate and do not limit theinvention.

Example 1 Detection of Exon 10 of the RhD Gene

A detection assay was performed to detect the exon10 region of theRhesus D gene. Design of PCR primers and detector oligonucleotide wasperformed according to the detailed description section. In thisparticular assay, the detector oligonucleotide carries anon-complementary 5′ overhang consisting of 6 Adenines. Since the targetsequence (Exon10 of RhD) was present in the sample, the PCR primers andthe detector oligonucleotide hybridized to the target. Duringamplification, the detector oligonucleotide was degraded by the 5′nuclease activity of the DNA polymerase extending from the upstream PCRprimer. During degradation, mass-distinguishable products (MDP's)including the 5′ polyA tag were released and identified unambiguously bymass spectrometric analysis (See FIG. 8). Detection of these masssignals confirmed the presence of the target nucleic acid.

Primer and Detector Oligonucleotide Sequences:

The following primers were used for amplification of a partial sequencewithin Exon 10 of the Rhesus D (RhD) gene:

Forward PCR primer: 5′ CCTCTCACTGTTGCCTGCATT 3′ (SEQ ID NO: 2)

Reverse PCR primer: 5′ AGTGCCTGCGCGAACATT 3′ (SEQ ID NO: 3)

The following detector oligonucleotide hybridized to the target and wasdegraded to yield MDP's that were detected by mass spectrometry:

Detector oligonucleotide: 5′ AAAAAAATTGCTGTCTGATCTTTATCCTCCGTTCCCT 3′(SEQ ID NO: 4)

PCR Mix:

PCR primers were used at a final concentration of 900 nM for the PCRprimers and at 200 nM of the detector oligonucleotide.

The PCR mix also contained 20 ng of genomic DNA (of an RhD+ individual),25 ul of 2×PCR Mastermix (ABI TaqMan®PCR master mix including buffer andAmpliTaq® Gold enzyme) and water to a final volume of 50 ul.

Reactions were performed in a 96-well ABGene microtiter plate.

Cycling Conditions:

The PCR mix was activated for 10 minutes at 95° C. and then subjected to40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute.

Sample Preparation for Mass Spectrometric Analysis:

10 ul of PCR product were transferred to a new 96-well microtiter plateand 20 ul of water containing 15 mg of ammonium-loaded ion-exchangeresin (Clean Resin, SEQUENOM®) were added to the PCR product. Thereaction mix was incubated for 15 minutes and gentle rotation.

A pintool device (Nanodispenser, SEQUENOM®) was used to transfer 15 nlof analyte on a miniaturized chip array (SpectroCHIP®, SEQUENOM®).

Mass Spectral Analysis:

Data acquisition and analysis were performed using a bench-top, linearMALDI-TOF mass spectrometer (Compact Analyzer, SEQUENOM®). For eachspectrum at least 20 laser shots were accumulated. Presence of thetarget nucleic acid (here Exon 10 of the RhD gene) was identified by themass-distinguishable products (MDP's) of the Exon10 specific detectoroligonucleotide. The MALDI-TOF MS spectrum exemplifies detection ofexon10 of the RhD gene (see FIG. 8). The three MDP's can only begenerated when the target sequence (Exon10 of the RhD gene) is presentduring amplification and when the detector oligonucleotide can hybridizeto the target nucleic acid during amplification.

Example 2 Detection of Exon 5 of the RhD Gene

A detection assay was performed to detect the exon5 region of the RhesusD gene. Design of PCR primers and detector oligonucleotide was performedaccording to the detailed description section. In this particular assay,the detector oligonucleotide carries a non-complementary 5′ overhangconsisting of 8 Adenines. Since the target sequence (Exon5 of RhD) waspresent in the sample, the PCR primers and the detector oligonucleotidehybridized to the target. During amplification, the detectoroligonucleotide was degraded by the 5′ nuclease activity of the DNApolymerase extending from the upstream PCR primer. During degradation,mass-distinguishable products (MDP's) including the 5′ polyA tag werereleased and identified unambiguously by mass spectrometric analysis(See FIG. 9). Detection of these mass signals confirmed the presence ofthe target nucleic acid.

Primer and Detector Oligonucleotide Sequences:

The following primers were used for amplification of a partial sequencewithin Exon 5 of the Rhesus D (RhD) gene:

Forward PCR primer: 5′ CGCCCTCTTCTTGTGGATG 3′ Reverse PCR primer:5′ GAACACGGCATTCTTCCTTTC 3′

The following detector oligonucleotide hybridized to the target and wasdegraded to yield MDP's that were detected by mass spectrometry:

Detector oligonucleotide: 5′ AAAAAAAATCTGGCCAAGTTTCAACTCTGCTCGCT 3′PCR Mix:

PCR primers were used at a final concentration of 900 nM for the PCRprimers and at 200 nM of the detector oligonucleotide.

The PCR mix also contained 20 ng of genomic DNA (of an RhD+ individual),25 ul of 2×PCR Mastermix (ABI TaqMan PCR master mix including buffer andAmpliTaq Gold enzyme) and water to a final volume of 50 ul.

Reactions were performed in a 96-well ABGene microtiter plate.

Cycling Conditions:

The PCR mix was activated for 10 minutes at 95° C. and then subjected to40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute.

Sample Preparation for Mass Spectrometric Analysis:

10 ul of PCR product were transferred to a new 96-well microtiter plateand 20 ul of water containing 15 mg of ammonium-loaded ion-exchangeresin (Clean Resin, SEQUENOM, Inc®) were added to the PCR product. Thereaction mix was incubated for 15 minutes and gentle rotation.

A pintool device (Nanodispenser, SEQUENOM, Inc®) was used to transfer 15nl of analyte on a miniaturized chip array (SpectroCHIP™, SEQUENOM,Inc®).

Mass Spectral Analysis:

Data acquisition and analysis were performed using a bench-top, linearMALDI-TOF mass spectrometer (Compact Analyzer, SEQUENOM, Inc®). For eachspectrum at least 20 laser shots were accumulated. Presence of thetarget nucleic acid (here Exon 5 of the RhD gene) was identified by themass-distinguishable products (MDP's) of the Exon5 specific detectoroligonucleotide. The MALDI-TOF MS spectrum exemplifies detection ofexon5 of the RhD gene (see FIG. 9). The three MDP's can only begenerated when the target sequence (Exon5 of the RhD gene) is presentduring amplification and when the detector oligonucleotide can hybridizeto the target nucleic acid during amplification.

Example 3 10-Plex Set of Y-Chromosome Markers for Gender DeterminationUsing Modified Detector Oligionucleotides that Contain LNAs and 3′Extension Blockers

A detection assay was performed to detect ten regions specific for theY-chromosome. PCR primers and detector oligonucleotides were designed tomeet the criteria described in the present invention using methods wellknown in the art. For example, the detector oligonucleotides weredesigned with a melting temperature approximately 10 degrees Celsiushigher than the PCR primers. Further, a polyA/G tail was added to the5′-end of the detector oligonucleotide with the length and sequencevariable in order to space and resolve cleavage products within a2000-6000 Da window on the MALDI-TOF MS.

In this particular multiplexed assay, the detector oligonucleotidescarry a non-complementary 5′-overhang consisting of multiple Adeninesand/or Guanines. In samples where the Y-chromosome is present (such asmale samples), the PCR primers and the detector oligonucleotideshybridize to the target. During amplification, the detectoroligonucleotides were degraded by the 5′-nuclease activity of the DNApolymerase extending from the upstream PCR primer. During degradation,nine of the ten assays were successful and mass-distinguishable products(MDP's) including the 5′-polyA or polyA/G tags were released andidentified unambiguously by MALDI-TOF mass spectrometric analysis.Detection of these mass signals confirmed the presence of the targetnucleic acid.

In samples where Y chromosome template is not present (such as femalesamples or negative control (NTC) samples), the PCR primers and thedetector oligonucleotides did not hybridize to the target, and 5′-polyAor polyA/G tags were not detected.

Primer and Detector Oligonucleotide Sequences

The detector oligonucleotides provided below hybridized to the targetand were degraded to yield MDP's that were detected by massspectrometry. The sequences may contain a “+”, which represents a lockednucleic acid (LNA), or “/3Phos/” and “/InvdT/” which represent theintroduction of a phosphate group and inverted deoxythymine,respectively.

Locked nucleic acids (LNAs) bind very stably with their complement andhave a highly reduced rate of cleavage relative to a nascentdeoxynucleotide. This serves to control the point of cleavage, andthereby produce uniform cleavage products. This effect may be furtherenhanced by placing two LNAs adjacent to each other.

The introduction of one or more phosphate groups or inverteddeoxythymines serves to block the 3′-end of the complementary portion ofthe detector oligonucleotide, which prevents extension of the detectoroligonucleotide by the DNA polymerase during cycling. Such unwantedextension can have several possible negative effects includingcompetitive binding for target after 5′-tail cleavage has occurred anddepletion of PCR reaction components such as dNTPs.

The following primers were used for amplification of a partial sequencewithin BPY2 gene (BPY2-2 assay):

Forward PCR primer: 5′-ACGTTGGATGATATTCTAGACTCTTCCAAGCC-3′ Reverse PCRprimer: 5′-ACGTTGGATGAAAAAGAGGAGTGTCACTCTAC-3′

Detector oligonucleotides (multiple detector oligonucleotides weretested individually in different assays):

5′-AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA-3′Where + represents a locked nucleic acid (LNA)

-   -   or

5′-AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA/3Phos/-3′ or5′-AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA/3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within CDY1 gene (CDY1-1 assay):

Forward PCR primer: 5′-ACGTTGGATGATGTTAGCCAGGATTGTCTCG-3′ Reverse PCRprimer: 5′-ACGTTGGATGACACCTGTAATCCCAGCATTTT-3′

Detector Oligonucleotides:

5′-AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA-3′ or5′-AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA/3Phos/-3′ or5′-AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA/3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin CDY1 gene (CDY1-2 assay):

Forward PCR primer: 5′-ACGTTGGATGCAATCCCGTGTCTTTCCT-3′ Reverse PCRprimer: 5′-ACGTTGGATGGAACCAAATACTGTGTATTCCC-3′

Detector Oligonucleotides:

5′-AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG-3′ or5′-AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG/3Phos/-3′ or5′-AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG/3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin CYORF14 region of the Y chromosome (CYORF14-3 assay):

Forward PCR primer: 5′-ACGTTGGATGTTTACATCAACAAACAAGGG-3′ Reverse PCRprimer: 5′-ACGTTGGATGCTACTGGGTCTAGCCTTATAAT-3′

Detector Oligonucleotides:

5′-AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT-3′ or5′-AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT/ 3Phos/-3′ or5′-AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT/ 3InvdT//-3′

The following primers were used for amplification of a partial sequencewithin PRY gene (PRY-2 assay):

Forward PCR primer: 5′-ACGTTGGATGTCACTGGGATCAGGACAGAC-3′ Reverse PCRprimer: 5′-ACGTTGGATGAGAGGAAACTGCTTCCCAAAC-3′

Detector Oligonucleotides:

5′-AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT-3′ or5′-AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT/3Phos/-3′ or5′-AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT/3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin RBMY1A1 gene (RBMY1A1-1 assay):

Forward PCR primer: 5′-ACGTTGGATGGATGGGTTTTCTATGTGTGGG-3′ Reverse PCRprimer: 5′-ACGTTGGATGTGAGTCTCTTAATAGCACTGAG-3′

Detector Oligonucleotides:

5′-AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA-3′ or5′-AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA/3Phos/-3′ or5′-AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA/3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within RBMY1A1 gene (RBMY1A1-2 assay):

Forward PCR primer: 5′-ACGTTGGATGAGCTAATTACTCATTTCCCCAG-3′ Reverse PCRprimer: 5′-ACGTTGGATGAGACTCAACAGGACAAGAGAC-3′

Detector Oligonucleotides:

5′-AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACCAA-3′ or5′-AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACCAA/ 3Phos/-3′ or5′-AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACCAA/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin RBMY2 gene (RBMY2-1 assay):

Forward PCR primer: 5′-ACGTTGGATGTGCAGAAAAGACCAAAGGAATC-3′ Reverse PCRprimer: 5′-ACGTTGGATGATAGATGCCACATAACTTGAGC-3′

Detector Oligonucleotides:

5′-AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC-3′ or5′-AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC/3Phos/-3′ or5′-AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC/3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin XKRY gene (XKRY-1 assay):

Forward PCR primer: 5′-ACGTTGGATGAACGTTTTACCGAAGTGTTGT-3′ Reverse PCRprimer: 5′-ACGTTGGATGAAGCCAAAGGCTAATATGTAGG-3′

Detector Oligonucleotides:

5′-AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTGA-3′ or5′-AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTGA/ 3Phos/-3′ or5′-AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTGA/ 3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within XKRY gene (XKRY-3 assay):

Forward PCR primer: 5′-ACGTTGGATGAGGCAAAATGTACTATGCCTAC-3′ Reverse PCRprimer: 5′-ACGTTGGATGTCCTGTAGTCTCAACTATTCAG-3′

Detector oligonucleotides:

5′-AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG-3′ or5′-AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG/3Phos/-3′ or5′-AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG/3InvdT/-3′PCR Mix:

PCR primers were used at a final concentration of 900 nM for the PCRprimers and at 250 nM of the detector oligonucleotide.

The PCR mix also contained 25 ng of genomic DNA (male or female), 25 ulof 2×PCR Mastermix (ABI TaqMan PCR master mix including buffer andAmpliTaq Gold enzyme) and water to a final volume of 50 ul.

Reactions were performed in a 96-well ABGene microtiter plate.

Cycling Conditions:

The PCR mix was activated for 10 minutes at 95° C. and then subjected to55 cycles of 95° C. for 30 seconds, 60° C. for 30 s and 72° C. for 1minute.

Sample Preparation for Mass Spectrometric Analysis:

10 ul of PCR product were transferred to a new 96-well microtiter plateand 20 ul of water containing 15 mg of ammonium-loaded ion-exchangeresin (Clean Resin, SEQUENOM, Inc®) were added to the PCR product. Thereaction mix was incubated for 15 minutes and gentle rotation.

A pintool device (Nanodispenser, SEQUENOM, Inc®) was used to transfer 15nl of analyte on a miniaturized chip array (SpectroCHIP™, SEQUENOM,Inc®).

Mass Spectral Analysis:

Data acquisition and analysis was performed using a bench-top, linearMALDI-TOF mass spectrometer (Compact Analyzer, SEQUENOM, Inc®). For eachspectrum at least 20 laser shots were accumulated. Presence of thetarget nucleic acid (here nine out of ten specific regions found on theY chromosome) was successfully identified by the mass-distinguishableproducts (MDP's) of the specific detector oligonucleotides per primerset. The MALDI-TOF MS spectrum exemplifies a 90% detection rate of a10-plex reaction. The ten MDP's are successfully generated when thetarget sequences (Y chromosome specific regions) are present duringamplification and when the detector oligonucleotides can hybridize tothe target nucleic acid during amplification.

Example 4 10-Plex Set of Y-Chromosome Markers for Gender DeterminationUsing 5′-Biotinylated Detector Oligonucleotides and Streptavidine-CoatedMagnetic Beads for Purification

A detection assay was performed to detect ten regions specific for theY-chromosome. The assay included an additional clean up step that usedbiotinylated detector oligonucleotides and streptavidine-coated magneticbeads for the capture of MDP's. PCR primers and detectoroligonucleotides were designed to meet the criteria described in thepresent invention using methods well known in the art. For example, thedetector oligonucleotides were designed with a melting temperatureapproximately 10 degrees Celsius higher than the PCR primers.

In this particular multiplexed assay, the detector oligonucleotidescarry a non-complementary 5′-overhang consisting of multiple Adeninesand/or Guanines. In samples where the Y-chromosome is present (such asmale samples), the PCR primers and the detector oligonucleotideshybridized to the target. During amplification, the detectoroligonucleotides were degraded by the 5′-nuclease activity of the DNApolymerase extending from the upstream PCR primer. During degradation,nine of the ten mass-distinguishable products (MDP's) including the5′-polyA or polyA/G tags were released and identified unambiguously byMALDI-TOF mass spectrometric analysis. Detection of these mass signalsconfirmed the presence of the target nucleic acid in nine of the tenassays.

In samples where Y chromosome template is not present (such as femalesamples or negative control (NTC) samples) the PCR primers and thedetector oligonucleotides do not hybridize to the target and 5′-polyA orpolyA/G tags are not detected. Uniplex reactions carried through theentire process and then pooled to detect all assays on a single chipelement also proved to be successful.

The detector oligonucleotides provided below hybridized to the targetand were degraded to yield MDP's that were detected by massspectrometry. The sequences may contain a “+”, which represents a lockednucleic acid (LNA), or “/3Phos/” and “/InvdT/” which represent theintroduction of a phosphate group and inverted deoxythymine,respectively. Also, the detector oligonucleotides may also contain a“Biosg/”, which represents a biotin.

The following primers were used for amplification of a partial sequencewithin BPY2 gene (BPY2-2 assay):

Forward PCR primer: 5′-ACGTTGGATGATATTCTAGACTCTTCCAAGCC-3′ Reverse PCRprimer: 5′-ACGTTGGATGAAAAAGAGGAGTGTCACTCTAC-3′

Detector oligonucleotides (multiple detector oligonucleotides weretested individually in different assays):

5′-5Biosg/AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA-3′ or5′-5Biosg/AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA/ 3Phos/-3′ or5′-5Biosg/AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA/ 3InvdT-3′

The following primers were used for amplification of a secondary partialsequence within CDY1 gene (CDY1-1 assay):

Forward PCR primer: 5′-ACGTTGGATGATGTTAGCCAGGATTGTCTCG-3′ Reverse PCRprimer: 5′-ACGTTGGATGACACCTGTAATCCCAGCATTTT-3′

Detector Oligonucleotides:

5′-5Biosg/AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA-3′ or5′-5Biosg/AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA/ 3Phos/-3′ or5′-5Biosg/AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin CDY1 gene (CDY1-2 assay):

Forward PCR primer: 5′-ACGTTGGATGCAATCCCGTGTCTTTCCT-3′ Reverse PCRprimer: 5′-ACGTTGGATGGAACCAAATACTGTGTATTCCC-3′

Detector Oligonucleotides:

5′-5Biosg/AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG-3′ or5′-5Biosg/AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG/ 3Phos/-3′ or5′-5Biosg/AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin CYORF14 region of the Y chromosome (CYORF14-3 assay):

Forward PCR primer: 5′-ACGTTGGATGTTTACATCAACAAACAAGGG-3′ Reverse PCRprimer: 5′-ACGTTGGATGCTACTGGGTCTAGCCTTATAAT-3′

Detector Oligonucleotides:

5′-5Biosg/AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGC T-3′ or5′-5Biosg/AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT/ 3Phos/-3′ or5′-5Biosg/AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin PRY gene (PRY-2 assay):

Forward PCR primer: 5′-ACGTTGGATGTCACTGGGATCAGGACAGAC-3′ Reverse PCRprimer: 5′-ACGTTGGATGAGAGGAAACTGCTTCCCAAAC-3′

Detector Oligonucleotides:

5′-5Biosg/AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT-3′ or5′-5Biosg/AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT/ 3Phos/-3′ or5′-5Biosg/AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin RBMY1A1 gene (RBMY1A1-1 assay):

Forward PCR primer: 5′-ACGTTGGATGGATGGGTTTTCTATGTGTGGG-3′ Reverse PCRprimer: 5′-ACGTTGGATGTGAGTCTCTTAATAGCACTGAG-3′

Detector Oligonucleotides:

5′-5Biosg/AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA-3′ or5′-5Biosg/AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA/ 3Phos/-3′ or5′-5Biosg/AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA/ 3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within RBMY1A1 gene (RBMY1A1-2 assay):

Forward PCR primer: 5′-ACGTTGGATGAGCTAATTACTCATTTCCCCAG-3′ Reverse PCRprimer: 5′-ACGTTGGATGAGACTCAACAGGACAAGAGAC-3′

Detector Oligonucleotides:

5′-5Biosg/AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACC AA-3′ or5′-5Biosg/AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACC AA/3Phos/-3′ or5′-5Biosg/AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACC AA/3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin RBMY2 gene (RBMY2-1 assay):

Forward PCR primer: 5′-ACGTTGGATGTGCAGAAAAGACCAAAGGAATC-3′ Reverse PCRprimer: 5′-ACGTTGGATGATAGATGCCACATAACTTGAGC-3′

Detector Oligonucleotides:

5′-/5Biosg/AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC-3′ or5′-/5Biosg/AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC/ 3Phos/-3′ or5′-/5Biosg/AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin XKRY gene (XKRY-1 assay):

Forward PCR primer: 5′-ACGTTGGATGAACGTTTTACCGAAGTGTTGT-3′ Reverse PCRprimer: 5′-ACGTTGGATGAAGCCAAAGGCTAATATGTAGG-3′

Detector Oligonucleotides:

5′-/5Biosg/AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTG A-3′ or5′-/5Biosg/AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTG A/3Phos/-3′ or5′-/5Biosg/AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTG A/3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within XKRY gene (XKRY-3 assay):

Forward PCR primer: 5′-ACGTTGGATGAGGCAAAATGTACTATGCCTAC-3′ Reverse PCRprimer: 5′-ACGTTGGATGTCCTGTAGTCTCAACTATTCAG-3′

Detector oligonucleotides:

5′-/5Biosg/AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG-3′ or5′-/5Biosg/AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG/ 3Phos/-3′ or5′-/5Biosg/AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG/ 3InvdT/-3′PCR Mix:

PCR primers were used at a final concentration of 900 nM for the PCRprimers and at 250 nM of the detector oligonucleotide.

The PCR mix also contained 25 ng of genomic DNA (male or female), 25 ulof 2×PCR Mastermix (ABI TaqMan PCR master mix including buffer andAmpliTaq Gold enzyme) and water to a final volume of 50 ul.

Reactions were performed in a 96-well ABGene microtiter plate.

Cycling Conditions:

The PCR mix was activated for 10 minutes at 95° C. and then subjected to55 cycles of 95° C. for 30 seconds, 60° C. for 30 s and 72° C. for 1minute.

Sample Preparation for Mass Spectrometric Analysis UsingStreptavidine-Coated Magnetic Beads (Invitrogen Corp®):

Bead Preparation:

-   -   1. Mix and aliquot 50 ul of beads-spin and spin and remove        supernatant    -   2. Add 75 ul of Wash Buffer (provided)-spin and remove        supernatant

Bead Binding:

-   -   3. Add 25 ul of Binding Buffer (provided) to beads    -   4. Add 35 ul of nanopure water to tube with beads    -   5. Add 15 ul of PCR/Nuclease reaction to tube with beads    -   6. Rotate 15 minutes at ambient temperature

Bead Washing:

-   -   7. Spin and remove supernatant until dry    -   8. Wash with 1× Wash Buffer (provided)    -   9. Spin and remove supernatant

Product Elution from Beads:

-   -   10. Add 25 ul of 25% NH₄OH (freshly prepared) to beads    -   11. Incubate for 10 minutes at 60° C.    -   12. Spin and remove supernatant to a new tube (contains product)    -   13. Shake with lid open for 60 minutes at ambient temperature

Optionally, Product Elution from Beads using Biotin Displacement ofBiotinylated Product:

-   -   10. Add 25 uls of free biotin dissolved in nanopure water (240        ng/ul). Mix to resuspend beads.    -   11. Incubate at 95° C. for 5 minutes followed by 4° C. hold.    -   12. Spin and remove supernatant to a new tube (contains product)

A pintool device (Nanodispenser, SEQUENOM, Inc®) was used to transfer 15nl of analyte on a miniaturized chip array (SpectroCHIP™, SEQUENOM,Inc®).

Mass Spectral Analysis:

Data acquisition and analysis were performed using a bench-top, linearMALDI-TOF mass spectrometer (Compact Analyzer, SEQUENOM, Inc®). For eachspectrum at least twenty laser shots were accumulated. Presence of thetarget nucleic acid (here nine out of ten specific regions found on theY chromosome) was identified by the mass-distinguishable products(MDP's) of the specific detector oligonucleotides per primer set. TheMALDI-TOF MS spectrum exemplifies a 90% detection rate of a 10-plexreaction. The nine MDP's were only generated when the target sequences(Y chromosome specific regions) were present during amplification andwhen the detector oligonucleotides hybridized to the target nucleic acidduring amplification. Uniplex amplification followed by pooling 5 ul ofeach reaction yielded similar results.

Example 5 10-Plex Set of Y-Chromosome Markers for Gender DeterminationUsing 5′-Photocleavable Biotin Modified Detector Oligonucleotides andStreotavidine-Coated Magnetic Beads for Purification

A detection assay was performed to detect ten regions specific for theY-chromosome. The assay included an additional clean up step that usedbiotinylated detector oligonucleotides and streptavidine-coated magneticbeads for the capture of MDP's. PCR primers and detectoroligonucleotides were designed to meet the criteria described in thepresent invention using methods well known in the art. For example, thedetector oligonucleotides were designed with a melting temperatureapproximately 10 degrees Celsius higher than the PCR primers.

In this particular multiplexed assay, the detector oligonucleotidescarry a non-complementary 5′-overhang consisting of multiple Adeninesand/or Guanines. In samples where the Y-chromosome is present (such asmale samples), the PCR primers and the detector oligonucleotideshybridized to the target. During amplification, the detectoroligonucleotides were degraded by the 5′-nuclease activity of the DNApolymerase extending from the upstream PCR primer. During degradation,eight of the ten mass-distinguishable products (MDP's) including the5′-polyA or polyA/G tags were released and identified unambiguously byMALDI-TOF mass spectrometric analysis. Detection of these mass signalsconfirmed the presence of the target nucleic acid in eight of the tenassays.

In samples where Y chromosome template is not present (such as femalesamples or negative control (NTC) samples) the PCR primers and thedetector oligonucleotides do not hybridize to the target and 5′-polyA orpolyA/G tags are not detected. Uniplex reactions carried through theentire process and then pooled to detect all assays on a single chipelement also proved to be successful.

The detector oligonucleotides provided below hybridized to the targetand were degraded to yield MDP's that were detected by massspectrometry. The sequences may contain a “+”, which represents a lockednucleic acid (LNA), or “/3Phos/” and “/InvdT/” which represent theintroduction of a phosphate group and inverted deoxythymine,respectively. Also, the detector oligonucleotides may also contain a“5PCBio”, which represents a photocleavable biotin.

The following primers were used for amplification of a partial sequencewithin BPY2 gene (BPY2-2 assay):

Forward PCR primer: 5-ACGTTGGATGATATTCTAGACTCTTCCAAGCC-3′ Reverse PCRprimer: 5′-ACGTTGGATGAAAAAGAGGAGTGTCACTCTAC-3′

Detector oligonucleotides (multiple detector oligonucleotides weretested individually in different assays):

5′-5PCBio/AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA-3′ or5′-5PCBio/AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA/ 3Phos/-3′ or5′-5PCBio/AAAAAAAAAAAAAT+T+TGCAAAGCCCAGCACTGA/ 3InvdT-3′

The following primers were used for amplification of a secondary partialsequence within CDY1 gene (CDY1-1 assay):

Forward PCR primer: 5′-ACGTTGGATGATGTTAGCCAGGATTGTCTCG-3′ Reverse PCRprimer: 5′-ACGTTGGATGACACCTGTAATCCCAGCATTTT-3′

Detector Oligonucleotides:

5′-5PCBio/AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA-3′ or5′-5PCBio/AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA/ 3Phos/-3′ or5′-5PCBio/AAAAAAAAAG+C+TGAGGTGCTTGGATCACGA/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin CDY1 gene (CDY1-2 assay):

Forward PCR primer: 5′-ACGTTGGATGCAATCCCGTGTCTTTCCT-3′ Reverse PCRprimer: 5′-ACGTTGGATGGAACCAAATACTGTGTATTCCC-3′

Detector Oligonucleotides:

5′-5PCBio/AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG-3′ or5′-5PCBio/AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG/ 3Phos/-3′ or5′-5PCBio/AAAAAAAAA+T+GGCTTCCCAGGAGTTTGAGG/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin CYORF14 region of the Y chromosome (CYORF14-3 assay):

Forward PCR primer: 5′-ACGTTGGATGTTTACATCAACAAACAAGGG-3′ Reverse PCRprimer: 5′-ACGTTGGATGCTACTGGGTCTAGCCTTATAAT-3′

Detector Oligonucleotides:

5′-5PCBio/AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGC T-3′ or5′-5PCBio/AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT/ 3Phos/-3′ or5′-5PCBio/AAAAGGAAAAAA+G+AGGTTGACATGAAGTCATTTGCT/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin PRY gene (PRY-2 assay):

Forward PCR primer: 5′-ACGTTGGATGTCACTGGGATCAGGACAGAC-3′ Reverse PCRprimer: 5′-ACGTTGGATGAGAGGAAACTGCTTCCCAAAC-3′

Detector Oligonucleotides:

5′-5PCBio/AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT-3′ or5′-5PCBio/AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT/ 3Phos/-3′ or5′-5PCBio/AAAAAAAAAAAAAAA+A+GCTGCCAGCAAGGAGCCT/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin RBMY1A1 gene (RBMY1A1-1 assay):

Forward PCR primer: 5′-ACGTTGGATGGATGGGTTTTCTATGTGTGGG-3′ Reverse PCRprimer: 5′-ACGTTGGATGTGAGTCTCTTAATAGCACTGAG-3′

Detector Oligonucleotides:

5′-5PCBio/AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA-3′ or5′-5PCBio/AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA/ 3Phos/-3′ or5′-5PCBio/AAAAAAAAAAA+C+GGGAGGAGTCAGTGGGGA/ 3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within RBMY1A1 gene (RBMY1A1-2 assay):

Forward PCR primer: 5′-ACGTTGGATGAGCTAATTACTCATTTCCCCAG-3′ Reverse PCRprimer: 5′-ACGTTGGATGAGACTCAACAGGACAAGAGAC-3′

Detector Oligonucleotides:

5′-5PCBio/AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACC AA-3′ or5′-5PCBio/AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACC AA/3Phos/-3′ or5′-5PCBio/AAAAAAAAAAAAAAAT+G+AGGACTTGTTTTGATTGAACC AA/3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin RBMY2 gene (RBMY2-1 assay):

Forward PCR primer: 5′-ACGTTGGATGTGCAGAAAAGACCAAAGGAATC-3′ Reverse PCRprimer: 5′-ACGTTGGATGATAGATGCCACATAACTTGAGC-3′

Detector Oligonucleotides:

5′-/5PCBio/AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC-3′ or5′-/5PCBio/AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC/ 3Phos/-3′ or5′-/5PCBio/AAAAAAAAAAAA+C+GAGGATCAGGGAGCACCC/ 3InvdT/-3′

The following primers were used for amplification of a partial sequencewithin XKRY gene (XKRY-1 assay):

Forward PCR primer: 5′-ACGTTGGATGAACGTTTTACCGAAGTGTTGT-3′ Reverse PCRprimer: 5′-ACGTTGGATGAAGCCAAAGGCTAATATGTAGG-3′

Detector Oligonucleotides:

5′-/5PCBio/AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATT GA-3′ or5′-/5PCBio/AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTG A/3Phos/-3′ or5′-/5PCBio/AAAAAAAAAAAAT+G+ATGAACTACACGGCAATTATTG A/3InvdT/-3′

The following primers were used for amplification of a secondary partialsequence within XKRY gene (XKRY-3 assay):

Forward PCR primer: 5′-ACGTTGGATGAGGCAAAATGTACTATGCCTAC-3′ Reverse PCRprimer: 5′-ACGTTGGATGTCCTGTAGTCTCAACTATTCAG-3′

Detector oligonucleotides:

5′-/5PCBio/AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG-3′ or5′-/5PCBio/AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG/ 3Phos/-3′ or5′-/5PCBio/AAAGGAAAAAA+T+GCTCACTTGGGCGAAGGAG/ 3InvdT/-3′PCR Mix:

PCR primers were used at a final concentration of 900 nM for the PCRprimers and at 250 nM of the detector oligonucleotide.

The PCR mix also contained 25 ng of genomic DNA (male or female), 25 ulof 2×PCR Mastermix (ABI TaqMan PCR master mix including buffer andAmpliTaq Gold enzyme) and water to a final volume of 50 ul.

Reactions were performed in a 96-well ABGene microtiter plate.

Cycling Conditions:

The PCR mix was activated for 10 minutes at 95° C. and then subjected to55 cycles of 95° C. for 30 seconds, 60° C. for 30 s and 72° C. for 1minute.

Sample Preparation for Mass Spectrometric Analysis UsingStreptavidine-Coated Magnetic Beads (Invitrogen Corp®):

Bead Preparation:

-   -   1. Mix and aliquot 50 ul of beads-spin and spin and remove        supernatant    -   2. Add 75 ul of Wash Buffer (provided)-spin and remove        supernatant

Bead Binding:

-   -   3. Add 25 ul of Binding Buffer (provided) to beads    -   4. Add 35 ul of nanopure water to tube with beads    -   5. Add 15 ul of PCR/Nuclease reaction to tube with beads    -   6. Rotate 15 minutes at ambient temperature

Bead Washing:

-   -   7. Spin and remove supernatant until dry    -   8. Wash with 1× Wash Buffer (provided)    -   9. Spin and remove supernatant

Product Elution from Beads Using Photocleavable Modified Probes:

-   -   10. Add 25 uls of nanopure water. Mix to resuspend beads.    -   11. Incubate under direct UV light (302-350 nm wavelength)        without plate seal for 5 minutes.    -   12. Spin and remove supernatant to a new tube (contains product)

A pintool device (Nanodispenser, SEQUENOM, Inc®) was used to transfer 15nl of analyte on a miniaturized chip array (SpectroCHIP™, SEQUENOM,Inc®).

Mass Spectral Analysis:

Data acquisition and analysis were performed using a bench-top, linearMALDI-TOF mass spectrometer (Compact Analyzer, SEQUENOM, Inc®). For eachspectrum at least twenty laser shots were accumulated. Presence of thetarget nucleic acid (here eight out of ten specific regions found on theY chromosome) was identified by the mass-distinguishable products(MDP's) of the specific detector oligonucleotides per primer set. TheMALDI-TOF MS spectrum exemplifies a 80% detection rate of a 10-plexreaction. The eight MDP's were only generated when the target sequences(Y chromosome specific regions) were present during amplification andwhen the detector oligonucleotides hybridized to the target nucleic acidduring amplification. Uniplex amplification followed by pooling 5 ul ofeach reaction yielded similar results.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the inventionclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a primer” can mean one or more primers)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value sometimes within 10% of the underlying parameter (i.e., plusor minus 10%), a value sometimes within 5% of the underlying parameter(i.e., plus or minus 5%), a value sometimes within 2.5% of theunderlying parameter (i.e., plus or minus 2.5%), or a value sometimeswithin 1% of the underlying parameter (i.e., plus or minus 1%), andsometimes refers to the parameter with no variation. For example, alength of “about 100 nucleotides” can include lengths between 90nucleotides and 110 nucleotides. Thus, it should be understood thatalthough the present invention has been specifically disclosed byrepresentative embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and such modifications and variations are consideredwithin the scope of this invention.

Embodiments of the invention are set forth in the claims that follow.

1. A detector oligonucleotide comprising a complementary portion that iscomplementary to a target nucleic acid, and a non-complementary portionthat is susceptible to cleavage by a cleavage agent, thereby producing amass distinguishable product (MDP), wherein the detector oligonucleotidecomprises a capture mechanism in the detector oligonucleotide on thenon-complementary portion that allows for the mass-distinguishableproduct to bind to a solid support upon its cleavage from the detectoroligonucleotide by the cleavage agent.
 2. The detector oligonucleotideof claim 1, further comprising a modification that increases the meltingtemperature of the detector oligonucleotide.
 3. The detectoroligonucleotide of claim 1, further comprising a modification thatincreases resistance of the detector oligonucleotide to cleavage.
 4. Thedetector oligonucleotide of claim 2, wherein the complementary portioncomprises a modification that increases the melting temperature of thedetector oligonucleotide.
 5. The detector oligonucleotide of claim 4,wherein the modification is the incorporation of one or more minorgroove binders to the detector oligonucleotide.
 6. The detectoroligonucleotide of claim 3, wherein the complementary portion comprisesa modification that increases resistance of the detector oligonucleotideto cleavage.
 7. The detector oligonucleotide of claim 6, wherein themodification is the incorporation of one or more locked nucleic acids(LNAs) or amadites.
 8. The detector oligonucleotide of claim 1, whereinthe capture mechanism is a biotin molecule.
 9. The detectoroligonucleotide of claim 1, wherein the mass-distinguishable product isamplified after its release from the detector oligonucleotide.
 10. Thedetector oligonucleotide of claim 9, wherein the detectoroligonucleotide comprises a universal primer hybridization site and isamplified using a universal primer.
 11. The detector oligonucleotide ofclaim 1, wherein the non-complementary portion comprises a detectionmoiety.
 12. The detector oligonucleotide of claim 1 that furthercomprises two or more of the modifications selected from the groupconsisting of minor groove binders, locked nucleic acids, amadites,capture mechanism, and detection moiety.
 13. A library comprising two ormore detector oligonucleotides of claim 1, wherein each massdistinguishable product corresponds to a unique target nucleic acid. 14.The library of claim 13 which comprises 10 or more detectoroligonucleotide species.
 15. The library of claim 13 which comprises 50or more detector oligonucleotide species.
 16. The library of claim 13which comprises 100 or more detector oligonucleotide species.
 17. Thelibrary of claim 13 which comprises 250 or more detector oligonucleotidespecies.